Polypeptide compounds for inhibiting angiogenesis and tumor growth

ABSTRACT

In certain embodiments, this present invention provides polypeptide compositions, including compositions containing a modified polypeptide, and methods for inhibiting Ephrin B2 or EphB4 activity. In other embodiments, the present invention provides methods and compositions for treating cancer or for treating angiogenesis-associated diseases.

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 60/612,488, filed Sep. 23, 2004, thespecification of which is incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION

Angiogenesis, the development of new blood vessels from the endotheliumof a preexisting vasculature, is a critical process in the growth,progression, and metastasis of solid tumors within the host. Duringphysiologically normal angiogenesis, the autocrine, paracrine, andamphicrine interactions of the vascular endothelium with its surroundingstromal components are tightly regulated both spatially and temporally.Additionally, the levels and activities of proangiogenic and angiostaticcytokines and growth factors are maintained in balance. In contrast, thepathological angiogenesis necessary for active tumor growth is sustainedand persistent, representing a dysregulation of the normal angiogenicsystem. Solid and hematopoietic tumor types are particularly associatedwith a high level of abnormal angiogenesis.

It is generally thought that the development of tumor consists ofsequential, and interrelated steps that lead to the generation of anautonomous clone with aggressive growth potential. These steps includesustained growth and unlimited self-renewal. Cell populations in a tumorare generally characterized by growth signal self-sufficiency, decreasedsensitivity to growth suppressive signals, and resistance to apoptosis.Genetic or cytogenetic events that initiate aberrant growth sustaincells in a prolonged “ready” state by preventing apoptosis.

It is a goal of the present disclosure to provide agents and therapeutictreatments for inhibiting angiogenesis and tumor growth.

SUMMARY OF THE INVENTION

In certain aspects, the disclosure provides polypeptide agents thatinhibit EphB4 or EphrinB2 mediated functions, including monomeric ligandbinding portions of the EphB4 and EphrinB2 proteins. As demonstratedherein, EphB4 and EphrinB2 participate in various disease states,including cancers and diseases related to unwanted or excessiveangiogenesis. Accordingly, certain polypeptide agents disclosed hereinmay be used to treat such diseases. In further aspects, the disclosurerelates to the discovery that EphB4 and/or EphrinB2 are expressed, oftenat high levels, in a variety of tumors. Therefore, polypeptide agentsthat down-regulate EphB4 or EphrinB2 function may affect tumors by adirect effect on the tumor cells as well as an indirect effect on theangiogenic processes recruited by the tumor. In certain embodiments, thedisclosure provides the identity of tumor types particularly suited totreatment with an agent that downregulates EphB4 or EphrinB2 function.In preferred embodiments, polypeptides disclosed herein are modified soas to have increased serum half-life in vivo.

In certain aspects, the disclosure provides soluble EphB4 polypeptidescomprising an amino acid sequence of an extracellular domain of an EphB4protein. The soluble EphB4 polypeptides bind specifically to an EphrinB2polypeptide. The term “soluble” is used merely to indicate that thesepolypeptides do not contain a transmembrane domain or a portion of atransmembrane domain sufficient to compromise the solubility of thepolypeptide in a physiological salt solution. Soluble polypeptides arepreferably prepared as monomers that compete with EphB4 for binding toligand such as EphrinB2 and inhibit the signaling that results fromEphB4 activation. Optionally, a soluble polypeptide may be prepared in amultimeric form, by, for example, expressing as an Fc fusion protein orfusion with another multimerization domain. Such multimeric forms mayhave complex activities, having agonistic or antagonistic effectsdepending on the context. In certain embodiments the soluble EphB4polypeptide comprises a globular domain of an EphB4 protein. A solubleEphB4 polypeptide may comprise a sequence at least 90% identical toresidues 1-522 of the amino acid sequence defined by FIG. 65 (SEQ IDNO:10). A soluble EphB4 polypeptide may comprise a sequence at least 90%identical to residues 1-412 of the amino acid sequence defined by FIG.65 (SEQ ID NO:10). A soluble EphB4 polypeptide may comprise a sequenceat least 90% identical to residues 1-312 of the amino acid sequencedefined by FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide maycomprise a sequence encompassing the globular (G) domain (amino acids29-197 of FIG. 65, SEQ ID NO:10), and optionally additional domains,such as the cysteine-rich domain (amino acids 239-321 of FIG. 65, SEQ IDNO:10), the first fibronectin type 3 domain (amino acids 324-429 of FIG.65, SEQ ID NO:10) and the second fibronectin type 3 domain (amino acids434-526 of FIG. 65, SEQ ID NO:10). Preferred polypeptides describedherein and demonstrated as having ligand binding activity includepolypeptides corresponding to 1-537, 1-427 and 1-326, respectively, ofthe amino acid sequence shown in FIG. 65 (SEQ ID NO:10). A soluble EphB4polypeptide may comprise a sequence as set forth in FIG. 1 or 2 (SEQ IDNos. 1 or 2). As is well known in the art, expression of such EphB4polypeptides in a suitable cell, such as HEK293T cell line, will resultin cleavage of a leader peptide. Although such cleavage is not alwayscomplete or perfectly consistent at a single site, it is known thatEphB4 tends to be cleaved so as to remove the first 15 amino acids ofthe sequence shown in FIG. 65 (SEQ ID NO:10). Accordingly, as specificexamples, the disclosure provides unprocessed soluble EphB4 polypeptidesthat bind to EphrinB2 and comprise an amino acid sequence selected fromthe following group (numbering is with respect to the sequence of FIG.65, SEQ ID NO:10): 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326,29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526,1-537 and 29-537. Additionally, heterologous leader peptides may besubstituted for the endogeneous leader sequences. Polypeptides may beused in a processed form, such forms having a predicted amino acidsequence selected from the following group (numbering is with respect tothe sequence of FIG. 65, SEQ ID NO:10): 16-197, 16-312, 16-321, 16-326,16-412, 16-427, 16-429, 16-526 and 16-537. Additionally, a soluble EphB4polypeptide may be one that comprises an amino acid sequence at least90%, and optionally 95% or 99% identical to any of the preceding aminoacid sequences while retaining EphrinB2 binding activity. Preferably,any variations in the amino acid sequence from the sequence shown inFIG. 65 (SEQ ID NO:10) are conservative changes or deletions of no morethan 1, 2, 3, 4 or 5 amino acids, particularly in a surface loop region.In certain embodiments, the soluble EphB4 polypeptide may inhibit theinteraction between Ephrin B2 and EphB4. The soluble EphB4 polypeptidemay inhibit clustering of or phosphorylation of Ephrin B2 or EphB4.Phosphorylation of EphrinB2 or EphB4 is generally considered to be oneof the initial events in triggering intracellular signaling pathwaysregulated by these proteins. As noted above, the soluble EphB4polypeptide may be prepared as a monomeric or multimeric fusion protein.The soluble polypeptide may include one or more modified amino acids.Such amino acids may contribute to desirable properties, such asincreased resistance to protease digestion.

The present disclosure provides soluble EphB4 polypeptides having anadditional component that confers increased serum half-life while stillretaining EphrinB2 binding activity. In certain embodiments solubleEphB4 polypeptides are monomeric and are covalently linked to one ormore polyoxyaklylene groups (e.g., polyethylene, polypropylene), andpreferably polyethylene glycol (PEG) groups. Accordingly, one aspect ofthe invention provides modified EphB4 polypeptides, wherein themodification comprises a single polyethylene glycol group covalentlybonded to the polypeptide. Other aspects provide modified EphB4polypeptides covalently bonded to one, two, three, or more polyethyleneglycol groups.

The one or more PEG may have a molecular weight ranging from about 1 kDato about 100 kDa, and will preferably have a molecular weight rangingfrom about 10 to about 60 kDa or about 10 to about 40 kDa. The PEG groupmay be a linear PEG or a branched PEG. In a preferred embodiment, thesoluble, monomeric EphB4 conjugate comprises an EphB4 polypeptidecovalently linked to one PEG group of from about 10 to about 40 kDa(monoPEGylated EphB4), or from about 15 to 30 kDa, preferably via anε-amino group of EphB4 lysine or the N-terminal amino group. Mostpreferably, EphB4 is randomly PEGylated at one amino group out of thegroup consisting of the ε-amino groups of EphB4 lysine and theN-terminal amino group.

In one embodiment, the pegylated polypeptides provided by the inventionhave a serum half-life in vivo at least 50%, 75%, 100%, 150% or 200%greater than that of an unmodified EphB4 polypeptide. In anotherembodiment, the pegylated EphB4 polypeptides provided by the inventioninhibit EphrinB2 activity. In a specific embodiment, they inhibitEphrinB2 receptor clustering, EphrinB2 phosphorylation, and/or EphrinB2kinase activity.

Surprisingly, it has been found that monoPEGylated EphB4 according tothe invention has superior properties in regard to the therapeuticapplicability of unmodified soluble EphB4 polypeptides andpoly-PEGylated EphB4. Nonetheless, the disclosure also providespoly-PEGylated EphB4 having PEG at more than one position. SuchpolyPEGylated forms provide improved serum-half life relative to theunmodified form.

In certain embodiments, a soluble EphB4 polypeptide is stably associatedwith a second stabilizing polypeptide that confers improved half-lifewithout substantially diminishing EphrinB2 binding. A stabilizingpolypeptide will preferably be immunocompatible with human patients (oranimal patients, where veterinary uses are contemplated) and have littleor no significant biological activity.

In a preferred embodiment, the stabilizing polypeptide is a human serumalbumin, or a portion thereof. A human serum albumin may be stablyassociated with the EphB4 polypeptide covalently or non-covalently.Covalent attachment may be achieved by expression of the EphB4polypeptide as a co-translational fusion with human serum albumin. Thealbumin sequence may be fused at the N-terminus, the C-terminus or at anon-disruptive internal position in the soluble EphB4 polypeptide.Exposed loops of the EphB4 would be appropriate positions for insertionof an albumin sequence. Albumin may also be post-translationallyattached to the EphB4 polypeptide by, for example, chemicalcross-linking. An EphB4 polypeptide may also be stably associated withmore than one albumin polypeptide. In some embodiments, the albumin isselected from the group consisting of a human serum albumin (HSA) andbovine serum albumin (BSA). In other embodiments, the albumin is anaturally occurring variant. In one preferred embodiment, the EphB4-HSAfusion inhibits the interaction between Ephrin B2 and EphB4, theclustering of Ephrin B2 or EphB4, the phosphorylation of Ephrin B2 orEphB4, or combinations thereof. In other embodiments, the EphB4-HSAfusion has enhanced in vivo stability relative to the unmodifiedwildtype polypeptide.

In certain aspects, the disclosure provides soluble EphrinB2polypeptides comprising an amino acid sequence of an extracellulardomain of an EphrinB2 protein. The soluble EphrinB2 polypeptides bindspecifically to an EphB4 polypeptide. The term “soluble” is used merelyto indicate that these polypeptides do not contain a transmembranedomain or a portion of a transmembrane domain sufficient to compromisethe solubility of the polypeptide in a physiological salt solution.Soluble polypeptides are preferably prepared as monomers that competewith EphrinB2 for binding to ligand such as EphB4 and inhibit thesignaling that results from EphrinB2 activation. Optionally, a solublepolypeptide may be prepared in a multimeric form, by, for example,expressing as an Fc fusion protein or fusion with anothermultimerization domain. Such multimeric forms may have complexactivities, having agonistic or antagonistic effects depending on thecontext. A soluble EphrinB2 polypeptide may comprise residues 1-225 ofthe amino acid sequence defined by FIG. 66 (SEQ ID NO:11). A solubleEphrinB2 polypeptide may comprise a sequence defined by FIG. 3. As iswell known in the art, expression of such EphrinB2 polypeptides in asuitable cell, such as HEK293T cell line, will result in cleavage of aleader peptide. Although such cleavage is not always complete orperfectly consistent at a single site, it is known that EphrinB2 tendsto be cleaved so as to remove the first 26 amino acids of the sequenceshown in FIG. 66 (SEQ ID NO:11). Accordingly, as specific examples, thedisclosure provides unprocessed soluble EphrinB2 polypeptides that bindto EphB4 and comprise an amino acid sequence corresponding to aminoacids 1-225 of FIG. 66 (SEQ ID NO:11). Such polypeptides may be used ina processed form, such forms having a predicted amino acid sequenceselected from the following group (numbering is with respect to thesequence of FIG. 66, SEQ ID NO:11): 26-225. In certain embodiments, thesoluble EphrinB2 polypeptide may inhibit the interaction between EphrinB2 and EphB4. The soluble EphrinB2 polypeptide may inhibit clustering ofor phosphorylation of EphrinB2 or EphB4. As noted above, the solubleEphrinB2 polypeptide may be prepared as a monomeric or multimeric fusionprotein. The soluble polypeptide may include one or more modified aminoacids. Such amino acids may contribute to desirable properties, such asincreased resistance to protease digestion.

In certain aspects, the disclosure provides pharmaceutical formulationscomprising a polypeptide reagent and a pharmaceutically acceptablecarrier. The polypeptide reagent may be any disclosed herein, including,for example, soluble EphB4 or EphrinB2 polypeptides. Additionalformulations include cosmetic compositions and diagnostic kits.

In certain aspects the disclosure provides methods of inhibitingsignaling through Ephrin B2/EphB4 pathway in a cell. A method maycomprise contacting the cell with an effective amount of a polypeptideagent, such as (a) a soluble polypeptide comprising an amino acidsequence of an extracellular domain of an EphB4 protein, wherein theEphB4 polypeptide is a monomer and binds specifically to an Ephrin B2polypeptide; (b) a soluble polypeptide comprising an amino acid sequenceof an extracellular domain of an Ephrin B2 protein, wherein the solubleEphrin B2 polypeptide is a monomer and binds with high affinity to anEphB4 polypeptide.

In certain aspects the disclosure provides methods for reducing thegrowth rate of a tumor, comprising administering an amount of apolypeptide agent sufficient to reduce the growth rate of the tumor. Thepolypeptide agent may be selected from the group consisting of: (a) asoluble polypeptide comprising an amino acid sequence of anextracellular domain of an EphB4 protein, wherein the EphB4 polypeptideis a monomer and binds specifically to an Ephrin B2 polypeptide, andoptionally comprises an additional modification to increase serumhalf-life, such as a PEGylation or serum albumin or both; (b) a solublepolypeptide comprising an amino acid sequence of an extracellular domainof an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is amonomer and binds with high affinity to an EphB4 polypeptide Optionally,the tumor comprises cells expressing a higher level of EphB4 and/orEphrinB2 than noncancerous cells of a comparable tissue.

In certain aspects, the disclosure provides methods for treating apatient suffering from a cancer. A method may comprise administering tothe patient a polypeptide agent. The polypeptide agent may be selectedfrom the group consisting of: (a) a soluble polypeptide comprising anamino acid sequence of an extracellular domain of an EphB4 protein,wherein the EphB4 polypeptide is a monomer and binds specifically to anEphrin B2 polypeptide, and optionally comprises an additionalmodification to increase serum half-life, such as a PEGylation or serumalbumin or both; (b) a soluble polypeptide comprising an amino acidsequence of an extracellular domain of an Ephrin B2 protein, wherein thesoluble Ephrin B2 polypeptide is a monomer and binds with high affinityto an EphB4 polypeptide. Optionally, the cancer comprises cancer cellsexpressing EphrinB2 and/or EphB4 at a higher level than noncancerouscells of a comparable tissue. The cancer may be a metastatic cancer. Thecancer may be selected from the group consisting of colon carcinoma,breast tumor, mesothelioma, prostate tumor, squamous cell carcinoma,Kaposi sarcoma, and leukemia. Optionally, the cancer is anangiogenesis-dependent cancer or an angiogenesis independent cancer. Thepolypeptide agent employed may inhibit clustering or phosphorylation ofEphrin B2 or EphB4. A polypeptide agent may be co-administered with oneor more additional anti-cancer chemotherapeutic agents that inhibitcancer cells in an additive or synergistic manner with the polypeptideagent.

In certain aspects, the disclosure provides methods of inhibitingangiogenesis. A method may comprise contacting a cell with an amount ofa polypeptide agent sufficient to inhibit angiogenesis. The polypeptideagent may be selected from the group consisting of: (a) a solublepolypeptide comprising an amino acid sequence of an extracellular domainof an EphB4 protein, wherein the EphB4 polypeptide is a monomer andbinds specifically to an Ephrin B2 polypeptide, and optionally comprisesan additional modification to increase serum half-life, such as aPEGylation or serum albumin or both; (b) a soluble polypeptidecomprising an amino acid sequence of an extracellular domain of anEphrin B2 protein, wherein the soluble Ephrin B2 polypeptide is amonomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for treating apatient suffering from an angiogenesis-associated disease, comprisingadministering to the patient a polypeptide agent. The polypeptide agentmay be selected from the group consisting of: (a) a soluble polypeptidecomprising an amino acid sequence of an extracellular domain of an EphB4protein, wherein the EphB4 polypeptide is a monomer and bindsspecifically to an Ephrin B2 polypeptide, and optionally comprises anadditional modification to increase serum half-life, such as aPEGylation or serum albumin or both; (b) a soluble polypeptidecomprising an amino acid sequence of an extracellular domain of anEphrin B2 protein, wherein the soluble Ephrin B2 polypeptide is amonomer and binds with high affinity to an EphB4 polypeptide. Thesoluble polypeptide may be formulated with a pharmaceutically acceptablecarrier. An angiogenesis related disease or unwanted angiogenesisrelated process may be selected from the group consisting ofangiogenesis-dependent cancer, benign tumors, inflammatory disorders,chronic articular rheumatism and psoriasis, ocular angiogenic diseases,Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma,telangiectasia psoriasis scleroderma, pyogenic granuloma, rubeosis,arthritis, diabetic neovascularization, vasculogenesis. A polypeptideagent may be co-administered with at least one additionalanti-angiogenesis agent that inhibits angiogenesis in an additive orsynergistic manner with the soluble polypeptide.

In certain aspects, the disclosure provides for the use of a polypeptideagent in the manufacture of medicament for the treatment of cancer or anangiogenesis related disorder. The polypeptide agent may be selectedfrom the group consisting of: (a) a soluble polypeptide comprising anamino acid sequence of an extracellular domain of an EphB4 protein,wherein the EphB4 polypeptide is a monomer and binds specifically to anEphrin B2 polypeptide, and optionally comprises an additionalmodification to increase serum half-life, such as a PEGylation or serumalbumin or both; (b) a soluble polypeptide comprising an amino acidsequence of an extracellular domain of an Ephrin B2 protein, wherein thesoluble Ephrin B2 polypeptide is a monomer and binds with high affinityto an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for treating apatient suffering from a cancer, comprising: (a) identifying in thepatient a tumor having a plurality of cancer cells that express EphB4and/or EphrinB2; and (b) administering to the patient a polypeptideagent. The polypeptide agent may be selected from the group consistingof: (i) a soluble polypeptide comprising an amino acid sequence of anextracellular domain of an EphB4 protein, wherein the EphB4 polypeptideis a monomer and binds specifically to an Ephrin B2 polypeptide, andoptionally comprises an additional modification to increase serumhalf-life, such as a PEGylation or serum albumin or both; (ii) a solublepolypeptide comprising an amino acid sequence of an extracellular domainof an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is amonomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for identifying atumor that is suitable for treatment with an EphrinB2 or EphB4antagonist. A method may comprise detecting in the tumor cell one ormore of the following characteristics: (a) expression of EphB4 proteinand/or mRNA; (b) expression of EphrinB2 protein and/or mRNA; (c) geneamplification (e.g., increased gene copy number) of the EphB4 gene; or(d) gene amplification of the EphrinB2 gene. A tumor cell having one ormore of characteristics (a)-(d) may be suitable for treatment with anEphrinB2 or EphB4 antagonist, such as a polypeptide agent describedherein.

Surprisingly, applicants have found that an EphB4 polypeptide lackingthe globular domain can in fact inhibit tumor growth in a xenograftmodel, inhibit angiogenic tube formation of vascular endothelial cellsand inhibit EphrinB2-activated autokinase activity of EphB4. While notwishing to be bound to any mechanism of action, it is expected that thepolypeptide either prevents EphB4 aggregation or stimulates theelimination (e.g. by endocytosis) of EphB4 from the plasma membrane.Accordingly, the disclosure provides isolated soluble polypeptidescomprising an amino acid sequence of a fibronectin type 3 domain of anEphB4 protein. Such polypeptides will preferably have a biologicaleffect, such as inhibiting an activity (e.g. aggregation or kinaseactivity) of an EphB4 or EphrinB2 protein, and particularly theinhibition of tumor growth in a human or in a mouse xenograft model ofcancer. Such polypeptides may also inhibit angiogenesis in vivo or in ancell-based assay system. Such polypeptides may not bind to EphrinB2 andmay specifically exclude all of or the functional (e.g., EphrinB2binding-) portions of the globular domain of an EphB4 protein. Such apolypeptide will preferably comprise amino acids corresponding to aminoacids 324-429 and/or 434-526 of the sequence of FIG. 65 (SEQ ID NO:10),or sequences at least 90%, 95%, 98%, 99% identical thereto. An exampleof such a polypeptide is shown in SEQ ID NO: 15. Such a polypeptide maybe modified in any of the ways described herein, and may be produced asa monomer or as a dimer or multimer comprising two or more suchpolypeptides, such as an Fc fusion construct. Dimers or multimers may bedesirable to enhance the effectiveness of such polypeptides. All of themethods for producing and using such polypeptides are similar to thosedescribed herein with respect to other EphB4 polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequence of the B4ECv3 protein (predictedsequence of the precursor including uncleaved Eph B4 leader peptide isshown; SEQ ID NO:1).

FIG. 2 shows amino acid sequence of the B4ECv3NT protein (predictedsequence of the precursor including uncleaved Eph B4 leader peptide isshown; SEQ ID NO:2).

FIG. 3 shows amino acid sequence of the B2EC protein (predicted sequenceof the precursor including uncleaved Ephrin B2 leader peptide is shown;SEQ ID NO:3).

FIG. 4 shows amino acid sequence of the B4ECv3-FC protein (predictedsequence of the precursor including uncleaved Eph B4 leader peptide isshown; SEQ ID NO:4).

FIG. 5 shows amino acid sequence of the B2EC-FC protein (predictedsequence of the precursor including uncleaved Ephrin B2 leader peptideis shown; SEQ ID NO:5).

FIG. 6 shows B4EC-FC binding assay (Protein A-agarose based).

FIG. 7 shows B4EC-FC inhibition assay (Inhibition in solution).

FIG. 8 shows B2EC-FC binding assay (Protein-A-agarose based assay).

FIG. 9 shows chemotaxis of HUAEC in response to B4Ecv3.

FIG. 10 shows chemotaxis of HHEC in response to B2EC-FC.

FIG. 11 shows chemotaxis of HHAEC in response to B2EC.

FIG. 12 shows effect of B4Ecv3 on HUAEC tubule formation.

FIG. 13 shows effect of B2EC-FC on HUAEC tubule formation.

FIG. 14 is a schematic representation of human Ephrin B2 constructs.

FIG. 15 is a schematic representation of human EphB4 constructs.

FIG. 16 shows the domain structure of the recombinant soluble EphB4ECproteins. Designation of the domains are as follows: L—leader peptide,G—globular (ligand-binding domain), C—Cys-rich domain, F1,F2—fibronectin type III repeats, H—6×His-tag.

FIG. 17 shows purification and ligand binding properties of the EphB4ECproteins. A. SDS-PAAG gel electrophoresis of purified EphB4-derivedrecombinant soluble proteins (Coomassie-stained). B. Binding of EphrinB2-AP fusion to EphB4-derived recombinant proteins immobilized onNi-NTA-agarose beads. Results of three independent experiments are shownfor each protein. Vertical axis—optical density at 420 nm.

FIG. 18 shows that EphB4v3 inhibits chemotaxis.

FIG. 19 shows that EphB4v3 inhibits tubule formation on Matrigel. Adisplays the strong inhibition of tubule formation by B4v3 in arepresentative experiment. B shows a quantitation of the reduction oftube-length obtained with B4v3 at increasing concentrations as well as areduction in the number of junctions, in comparison to cells with noprotein. Results are displayed as mean values±S.D. obtained from threeindependent experiments performed with duplicate wells.

FIG. 20 shows that soluble EphB4 has no detectable cytotoxic effect asassessed by MTS assay.

FIG. 21 shows that B4v3 inhibits invasion and tubule formation byendothelial cells in the Matrigel assay. (A) to detect total invadingcells, photographed at 20× magnification or with Masson's Trichrome Topleft of A B displays section of a Matrigel plug with no GF, top right ofA displays section with B4IgG containing GF and lower left sectioncontains GF, and lower right shows GF in the presence of B4v3.Significant invasion of endothelial cells is only seen in GF containingMatrigel. Top right displays an area with a high number of invaded cellsinduced by B4IgG, which signifies the dimeric form of B4v3. The leftupper parts of the pictures correspond to the cell layers formed aroundthe Matrigel plug from which cells invade toward the center of the pluglocated in the direction of the right lower corner. Total cells insections of the Matrigel plugs were quantitated with Scion Imagesoftware. Results obtained from two experiments with duplicate plugs aredisplayed as mean values±S.D.

FIG. 22 shows tyrosine phosphorylation of EphB4 receptor in PC3 cells inresponse to stimulation with EphrinB2-Fc fusion in presence or absenceof EphB4-derived recombinant soluble proteins.

FIG. 23 shows effects of soluble EphB4ECD on viability and cell cycle.A) 3-day cell viability assay of two HNSCC cell lines. B) FACS analysisof cell cycle in HNSCC-15 cells treated as in A. Treatment of thesecells resulted in accumulation in subG0/G1 and S/G2 phases as indicatedby the arrows.

FIG. 24 shows that B4v3 inhibits endovascular response in a murinecorneal hydron micropocket assay.

FIG. 25 shows that that SCC15, B16, and MCF-7 co-injected with sB4v3 inthe presence of matrigel and growth factors, inhibits the in vivo tumorgrowth of these cells.

FIG. 26 shows that soluble EphB4 causes apoptosis, necrosis anddecreased angiogenesis in three tumor types, B16 (melanoma), SCC15 (headand neck carcinoma), and MCF-7 (breast carcinoma). Tumors were injectedpremixed with Matrigel plus growth factors and soluble EphB4subcutaneously. After 10 to 14 days, the mice were injectedintravenously with FITC-lectin (green) to assess blood vessel perfusion.Tumors treated with control PBS displayed abundant tumor density and arobust angiogenic response. Tumors treated with sEphB4 displayed adecrease in tumor cell density and a marked inhibition of tumorangiogenesis in regions with viable tumor cells, as well as tumornecrosis and apoptosis.

FIG. 27 shows expression of EphB4 in prostate cell lines. A) Westernblot of total cell lysates of various prostate cancer cell lines, normalprostate gland derived cell line (MLC) and acute myeloblastic lymphomacells (AML) probed with EphB4 monoclonal antibody. B) Phosphorylation ofEphB4 in PC-3 cells determined by Western blot.

FIG. 28 shows expression of EphB4 in prostate cancer tissue.Representative prostate cancer frozen section stained with EphB4monoclonal antibody (top left) or isotype specific control (bottomleft). Adjacent BPH tissue stained with EphB4 monoclonal antibody (topright). Positive signal is brown color in the tumor cells. Stroma andthe normal epithelia are negative. Note membrane localization of stainin the tumor tissue, consistent with trans-membrane localization ofEphB4. Representative QRT-PCR of RNA extracted from cancer specimens andadjacent BPH tissues (lower right).

FIG. 29 shows downregulation of EphB4 in prostate cancer cells by tumorsuppressors and RXR expression. A) PC3 cells were co-transfected withtruncated CD4 and p53 or PTEN or vector only. 24 h later CD4-sortedcells were collected, lysed and analyzed sequentially by Western blotfor the expression of EphB4 and β-actin, as a normalizer protein. B)Western blot as in (A) of various stable cell lines. LNCaP-FGF is astable transfection clone of FGF-8, while CWR22R-RXR stably expressesthe RXR receptor. BPH-1 was established from benign hypertrophicprostatic epithelium.

FIG. 30 shows regulation of EphB4 in prostate cancer cells by EGFR andIGFR-1. A) Western blot of PC3 cells treated with or without EGFRspecific inhibitor AG1478 (1 nM) for 36 hours. Decreased EphB4 signal isobserved after AG 1478 treatment. The membrane was stripped and reprobedwith β-actin, which was unaffected. B) Western Blot of triplicatesamples of PC3 cells treated with or without IGFR-1 specificneutralizing antibody MAB391 (2 μg/ml; overnight). The membrane wassequentially probed with EphB4, IGFR-1 and β-actin antibodies. IGFR-1signal shows the expected repression of signal with MAB391 treatment.

FIG. 31 shows effect of specific EphB4 AS-ODNs and siRNA on expressionand prostate cell functions. A) 293 cells stably expressing full-lengthconstruct of EphB4 was used to evaluate the ability of siRNA 472 toinhibit EphB4 expression. Cells were transfected with 50 nM RNAi usingLipofectamine 2000. Western blot of cell lysates 40 h post transfectionwith control siRNA (green fluorescence protein; GFP siRNA) or EphB4siRNA 472, probed with EphB4 monoclonal antibody, stripped and reprobedwith β-actin monoclonal antibody. B) Effect of EphB4 AS-10 on expressionin 293 transiently expressing full-length EphB4. Cells were exposed toAS-10 or sense ODN for 6 hours and analyzed by Western blot as in (A).C) 48 h viability assay of PC3 cells treated with siRNA as described inthe Methods section. Shown is mean±s.e.m. of triplicate samples. D)5-day viability assay of PC3 cells treated with ODNs as described in theMethods. Shown is mean±s.e.m. of triplicate samples. E) Scrape assay ofmigration of PC3 cells in the presence of 50 nM siRNAs transfected as in(A). Shown are photomicrographs of representative 20× fields takenimmediately after the scrape was made in the monolayer (0 h) and after20 h continued culture. A large number of cells have filled in thescrape after 20 h with control siRNA, but not with EphB4 siRNA 472. F)Shown is a similar assay for cells treated with AS-10 or sense ODN (both10 μM). G) Matrigel invasion assay of PC3 cells transfected with siRNAor control siRNA as described in the methods. Cells migrating to theunderside of the Matrigel coated insert in response to 5 mg/mlfibronectin in the lower chamber were fixed and stained with Giemsa.Shown are representative photomicrographs of control siRNA and siRNA 472treated cells. Cell numbers were counted in 5 individual high-poweredfields and the average±s.e.m. is shown in the graph (bottom right).

FIG. 32 shows effect of EphB4 siRNA 472 on cell cycle and apoptosis. A)PC3 cells transfected with siRNAs as indicated were analyzed 24 h posttransfection for cell cycle status by flow cytometry as described in theMethods. Shown are the plots of cell number vs. propidium iodidefluorescence intensity. 7.9% of the cell population is apoptotic (in theSub G0 peak) when treated with siRNA 472 compared to 1% with controlsiRNA. B) Apoptosis of PC3 cells detected by Cell Death DetectionELISA^(plus) kit as described in the Methods. Absorbance at 405 nmincreases in proportion to the amount of histone and DNA-POD in thenuclei-free cell fraction. Shown is the mean±s.e.m. of triplicatesamples at the indicated concentrations of siRNA 472 and GFP siRNA(control).

FIG. 33 shows that EphB4 and EphrinB2 are expressed in mesothelioma celllines as shown by RT-PCR (A) and Western Blot (B).

FIG. 34 shows expression of ephrin B2 and EphB4 by in situ hybridizationin mesothelioma cells. NCI H28 mesothelioma cell lines cultured inchamber slides hybridized with antisense probe to ephrin B2 or EphB4(top row). Control for each hybridization was sense (bottom row).Positive reaction is dark blue cytoplasmic stain.

FIG. 35 shows cellular expression of EphB4 and ephrin B2 in mesotheliomacultures. Immunofluorescence staining of primary cell isolate derivedfrom pleural effusion of a patient with malignant mesothelioma and celllines NCI H28, NCI H2373, and NCI H2052 for ephrin B2 and EphB4. Greencolor is positive signal for FITC labeled secondary antibody.Specificity of immunofluorescence staining was demonstrated by lack ofsignal with no primary antibody (first row). Cell nuclei werecounterstained with DAPI (blue color) to reveal location of all cells.Shown are merged images of DAPI and FITC fluorescence. Originalmagnification 200×.

FIG. 36 shows expression of ephrin B2 and EphB4 in mesothelioma tumor.Immunohistochemistry of malignant mesothelioma biopsy. H&E stainedsection reveals tumor architecture; bottom left panel is backgroundcontrol with no primary antibody. EphB4 and ephrin B2 specific stainingis brown color. Original magnification 200×.

FIG. 37 shows effects of EPHB4 antisense probes (A) and EPHB4 siRNAs (B)on the growth of H28 cells.

FIG. 38 shows effects of EPHB4 antisense probes (A) and EPHB4 siRNAs (B)on cell migration.

FIG. 39 shows that EphB4 is expressed in HNSCC primary tissues andmetastases. A) Top: Immunohistochemistry of a representative archivalsection stained with EphB4 monoclonal antibody as described in themethods and visualized with DAB (brown color) localized to tumor cells.Bottom: Hematoxylin and Eosin (H&E) stain of an adjacent section. Densepurple staining indicates the presence of tumor cells. The right handcolumn are frozen sections of lymph node metastasis stained with EphB4polyclonal antibody (top right) and visualized with DAB. Control(middle) was incubation with goat serum and H&E (bottom) reveals thelocation of the metastatic foci surrounded by stroma which does notstain. B) In situ hybridization of serial frozen sections of a HNSCCcase probed with EphB4 (left column) and ephrin B2 (right column) DIGlabeled antisense or sense probes generated by run-off transcription.Hybridization signal (dark blue) was detected usingalkaline-phosphatase-conjugated anti-DIG antibodies and sections werecounterstained with Nuclear Fast Red. A serial section stained with H&Eis shown (bottom left) to illustrate tumor architecture. C) Western blotof protein extract of patient samples consisting of tumor (T),uninvolved normal tissue (N) and lymph node biopsies (LN). Samples werefractionated by polyacrylamide gel electrophoresis in 4-20% Tris-glycinegels and subsequently electroblotted onto nylon membranes. Membraneswere sequentially probed with EphB4 monoclonal antibody and β-actinMoAb. Chemiluminescent signal was detected on autoradiography film.Shown is the EphB4 specific band which migrated at 120 kD and β-actinwhich migrated at 40 kD. The β-actin signal was used to control forloading and transfer of each sample.

FIG. 40 shows that EphB4 is expressed in HNSCC cell lines and isregulated by EGF: A) Survey of EphB4 expression in SCC cell lines.Western blot of total cell lysates sequentially probed with EphB4monoclonal antibody, stripped and reprobed with β-actin monoclonalantibody as described for FIG. 39C. B) Effect of the specific EGFRinhibitor AG1478 on EphB4 expression: Western blot of crude cell lysatesof SCC15 treated with 0-1000 nM AG 1478 for 24 h in media supplementedwith 10% FCS (left) or with 1 mM AG 1478 for 4, 8, 12 or 24 h (right).Shown are membranes sequentially probed for EphB4 and β-actin. C) Effectof inhibition of EGFR signaling on EphB4 expression in SCC cell lines:Cells maintained in growth media containing 10% FCS were treated for 24hr with 1 μM AG 1478, after which crude cell lysates were analyzed byWestern blots of cell lysates sequentially probed with for EGFR, EphB4,ephrin B2 and β-actin antibodies. Specific signal for EGFR was detectedat 170 kD and ephrin B2 at 37 kD in addition to EphB4 and β-actin asdescribed in FIG. 1C. β-actin serves as loading and transfer control.

FIG. 41 shows mechanism of regulation of EphB4 by EGF: A) Schematic ofthe EGFR signaling pathways, showing in red the sites of action andnames of specific kinase inhibitors used. B) SCC15 cells wereserum-starved for 24 h prior to an additional 24 incubation as indicatedwith or without EGF (10 ng/ml), 3 μM U73122, or 5 μM SH-5, 5 μMSP600125, 25 nM LY294002, —μM PD098095 or 5 μM SB203580. N/A indicatescultures that received equal volume of diluent (DMSO) only. Cell lysateswere subjected to Western Blot with EphB4 monoclonal antibody. β-actinsignal serves as control of protein loading and transfer.

FIG. 42 shows that specific EphB4 siRNAs inhibit EphB4 expression, cellviability and cause cell cycle arrest. A) 293 cells stably expressingfull length EphB4 were transfected with 50 nM RNAi usingLipofectamine™2000. 40 h post-transfection cells were harvested, lysedand processed for Western blot. Membranes were probed with EphB4monoclonal antibody, stripped and reprobed with β-actin monoclonalantibody as control for protein loading and transfer. Negative reagentcontrol was RNAi to scrambled green fluorescence protein (GFP) sequenceand control is transfection with Lipofectamine™2000 alone. B) MTT cellviability assays of SCC cell lines treated with siRNAs for 48 h asdescribed in the Methods section. Shown is mean±s.e.m. of triplicatesamples. C) SCC15 cells transfected with siRNAs as indicated wereanalyzed 24 h post transfection for cell cycle status by flow cytometryas described in the Methods. Shown are the plots of cell number vs.propidium iodide fluorescence intensity. Top and middle row show plotsfor cells 16 h after siRNA transfection, bottom row shows plots forcells 36 h post transfection. Specific siRNA and concentration areindicated for each plot. Lipo=Lipofectamine™200 mock transfection.

FIG. 43 shows in vitro effects of specific EphB4 AS-ODNs on SCC cells.A) 293 cells transiently transfected with EphB4 full-length expressionplasmid were treated 6 h post transfection with antisense ODNs asindicated. Cell lysates were collected 24 h after AS-ODN treatment andsubjected to Western Blot. B) SCC25 cells were seeded on 48 well platesat equal densities and treated with EphB4 AS-ODNs at 1, 5, and 10 μM ondays 2 and 4. Cell viability was measured by MTT assay on day 5. Shownis the mean±s.e.m. of triplicate samples. Note that AS-ODNs that wereactive in inhibiting EphB4 protein levels were also effective inhibitorsof SCC15 cell viability. C) Cell cycle analysis of SCC15 cells treatedfor 36 h with AS-10 (bottom) compared to cells that were not treated(top). D) Confluent cultures of SCC15 cells scraped with a plasticPasteur pipette to produce 3 mm wide breaks in the monolayer. Theability of the cells to migrate and close the wound in the presence ofinhibiting EphB4 AS-ODN (AS-10) and non-inhibiting AS-ODN (AS-1) wasassessed after 48 h. Scrambled ODN is included as a negative controlODN. Culture labeled no treatment was not exposed to ODN. At initiationof the experiment, all cultures showed scrapes of equal width andsimilar to that seen in 1 μM EphB4 AS-10 after 48 h. The red bracketsindicate the width of the original scrape. E) Migration of SCC15 cellsin response to 20 mg/ml EGF in two-chamber assay as described in theMethods. Shown are representative photomicrographs of non-treated (NT),AS-6 and AS-10 treated cells and 10 ng/ml Taxol as positive control ofmigration inhibition. F) Cell numbers were counted in 5 individualhigh-powered fields and the average+s.e.m. is shown in the graph.

FIG. 44 shows that EphB4 AS-ODN inhibits tumor growth in vivo. Growthcurves for SCC15 subcutaneous tumor xenografts in Balb/C nude micetreated with EphB4 AS-10 or scrambled ODN at 20 mg/kg/day starting theday following implantation of 5×106 cells. Control mice received andequal volume of diluent (PBS). Shown are the mean±s.e.m. of 6mice/group. *P=0.0001 by Student's t-test compared to scrambled ODNtreated group.

FIG. 45 shows that Ephrin B2, but not EphB4 is expressed in KS biopsytissue. (A) In situ hybridization with antisense probes for ephrin B2and EphB4 with corresponding H&E stained section to show tumorarchitecture. Dark blue color in the ISH indicates positive reaction forephrin B2. No signal for EphB4 was detected in the Kaposi's sarcomabiopsy. For contrast, ISH signal for EphB4 is strong in squamous cellcarcinoma tumor cells. Ephrin B2 was also detected in KS using EphB4-APfusion protein (bottom left). (B) Detection of ephrin B2 with EphB4/Fcfusion protein. Adjacent sections were stained with H&E (left) to showtumor architecture, black rectangle indicates the area shown in theEphB4/Fc treated section (middle) detected with FITC-labeled anti-humanFc antibody as described in the methods section. As a control anadjacent section was treated with human Fc fragment (right). Specificsignal arising from EphB4/Fc binding to the section is seen only inareas of tumor cells. (C) Co-expression of ephrin B2 and the HHV8latency protein LANA1. Double-label confocal immunofluorescencemicroscopy with antibodies to ephrin B2 (red) LANA1 (green), or EphB4(red) of frozen KS biopsy material directly demonstrates co-expressionof LANA1 and ephrin B2 in KS biopsy. Coexpression is seen as yellowcolor. Double label confocal image of biopsy with antibodies to PECAM-1(green) in cells with nuclear propidium iodide stain (red),demonstrating the vascular nature of the tumor.

FIG. 46 shows that HHV-8 induces arterial marker expression in venousendothelial cells. (A) Immunofluorescence of cultures of HUVEC andHUVEC/BC-1 for artery/vein markers and viral proteins. Cultures weregrown on chamber slides and processed for immunofluorescence detectionof ephrin B2 (a, e, i), EphB4 (m, q, u), CD148 (j, v), and the HHV-8proteins LANA1 (b, f, m) or ORF59 (r) as described in the Materials andMethods. Yellow color in the merged images of the same field demonstrateco-expression of ephrin B2 and LANA or ephrin B2 and CD148. Thepositions of viable cells were revealed by nuclear staining with DAPI(blue) in the third column (c, g, k, o, s, w). Photomicrographs are ofrepresentative fields. (B) RT-PCR of HUVEC and two HHV-8 infectedcultures (HUVEC/BC-1 and HUVEC/BC-3) for ephrin B2 and EphB4. Ephrin B2product (200 bp) is seen in HUVEC/BC-1, HUVEC/BC-3 and EphB4 product(400 bp) is seen in HUVEC. Shown also is β-actin RT-PCR as a control foramount and integrity of input RNA.

FIG. 47 shows that HHV-8 induces arterial marker expression in Kaposi'ssarcoma cells. (A) Western blot for ephrin B2 on various cell lysates.SLK-vGPCR is a stable clone of SLK expressing the HHV-8 vGPCR, andSLK-pCEFL is control stable clone transfected with empty expressionvector. SLK cells transfected with LANA or LANAΔ440 are SLK-LANA andSLK-Δ440 respectively. Quantity of protein loading and transfer wasdetermined by reprobing the membranes with β-actin monoclonal antibody.(B) Transient transfection of KS-SLK cells with expression vectorpvGPCR-CEFL resulted in the expression of ephrin B2 as shown byimmunofluorescence staining with FITC (green), whereas the controlvector pCEFL had no effect. KS-SLK cells (0.8×105/well) were transfectedwith 0.8 μg DNA using Lipofectamine 2000. 24 hr later cells were fixedand stained with ephrin B2 polyclonal antibody and FITC conjugatedsecondary antibody as described in the methods. (C) Transienttransfection of HUVEC with vGPCR induces transcription from ephrin B2luciferase constructs. 8×103 HUVEC in 24 well plates were transfectedusing Superfect with 0.8 μg/well ephrin B2 promoter constructscontaining sequences from −2941 to −11 with respect to the translationstart site, or two 5′-deletions as indicated, together with 80 ng/wellpCEFL or pvGPCR-CEFL. Luciferase was determined 48 h post transfectionand induction ratios are shown to the right of the graph. pGL3Basic ispromoterless luciferase control vector. Luciferase was normalized toprotein since GPCR induced expression of the cotransfectedβ-galactosidase. Graphed is mean±SEM of 6 replicates. Shown is one ofthree similar experiments.

FIG. 48 shows that VEGF and VEGF-C regulate ephrin B2 expression. A)Inhibition of ephrin B2 by neutralizing antibodies. Cells were culturedin full growth medium and exposed to antibody (100 ng/ml) for 36 hrbefore collection and lysis for Western blot. B) For induction of ephrinB2 expression cells were cultured in EBM growth medium containing 5%serum lacking growth factors. Individual growth factors were added asindicated and the cells harvested after 36 h. Quantity of proteinloading and transfer was determined by reprobing the membranes β-actinmonoclonal antibody.

FIG. 49 shows that Ephrin B2 knock-down with specific siRNA inhibitsviability in KS cells and HUVEC grown in the presence of VEGF but notIGF, EGF or bFGF. A) KS-SLK cells were transfected with various siRNA toephrin B2 and controls. After 48 hr the cells were harvested and crudecell lysates fractionated on 4-20% SDS-PAGE. Western blot was performedwith monoclonal antibody to ephrin B2 generated in-house. The membranewas stripped and reprobed with β-actin monoclonal antibody (Sigma) toillustrate equivalent loading and transfer. B) 3 day cell viabilityassay of KS-SLK cultures in the presence of ephrin B2 and EphB4 siRNAs.1×10⁵ cells/well in 24-well plates were treated with 0, 10 and 100 ng/mlsiRNAs as indicated on the graph. Viability of cultures was determinedby MTT assay as described in the methods section. Shown are themean+standard deviation of duplicate samples. C) HUVE cells were seededon eight wells chamber slides coated with fibronectin. The HUVE cellswere grown overnight in EGM-2 media, which contains all growthsupplements. On the following day, the media was replaced with mediacontaining VEGF (10 ng/ml) or EGF, FGF and IGF as indicated. After 2 hrsof incubation at 37° C., the cells were transfected using Lipofectamine2000 (Invitrogen) in Opti-MEM medium containing 10 nM of siRNA to ephrinB2, Eph B4 or green fluorescence protein (GFP) as control. The cellswere incubated for 2 hr and then the fresh media containing growthfactors or VEGF alone was added to their respective wells. After 48 hrs,the cells were stained with crystal violet and the pictures were takenimmediately by digital camera at 10× magnification.

FIG. 50 shows that soluble EphB4 inhibits KS and EC cord formation andin vivo angiogenesis. Cord formation assay of HUVEC in Matrigel™ (upperrow). Cells in exponential growth phase were treated overnight with theindicated concentrations of EphB4 extracellular domain (ECD) prior toplating on Matrigel™. Cells were trypsinized and plated (1×10⁵cells/well) in a 24-well plate containing 0.5 ml Matrigel™. Shown arerepresentative 20× phase contrast fields of cord formation after 8 hrplating on Matrigel™ in the continued presence of the test compounds asshown. Original magnification 200×. KS-SLK cells treated in a similarmanner (middle row) in a cord formation assay on Matrigel™. Bottom rowshows in vivo Matrigel™ assay: Matrigel™ plugs containing growth factorsand EphB4 ECD or PBS were implanted subcutaneously in the mid-ventralregion of mice. After 7 days the plugs were removed, sectioned andstained with H&E to visualize cells migrating into the matrix. Intactvessels with large lumens are observed in the control, whereas EphB4 ECDalmost completely inhibited migration of cells into the Matrigel.

FIG. 51 shows expression of EPHB4 in bladder cancer cell lines (A), andregulation of EPHB4 expression by EGFR signaling pathway (B).

FIG. 52 shows that transfection of p53 inhibit the expression of EPHB4in 5637 cell.

FIG. 53 shows growth inhibition of bladder cancer cell line (5637) upontreatment with EPHB4 siRNA 472.

FIG. 54 shows results on apoptosis study of 5637 cells transfected withEPHB4 siRNA 472.

FIG. 55 shows effects of EPHB4 antisense probes on cell migration. 5637cells were treated with EPHB4AS10 (10 μM) (bottom panels). Upper panelsshow control cells.

FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637 cells weretransfected with siRNA 472 or control siRNA.

FIG. 57 shows comparison of EphB4 monoclonal antibodies by G250 and inpull-down assay.

FIG. 58 shows that EphB4 antibodies inhibit the growth of SCC15xenograft tumors.

FIG. 59 shows that EphB4 antibodies cause apoptosis, necrosis anddecreased angiogenesis in SCC15, head and neck carcinoma tumor type.

FIG. 60 shows that systemic administration of EphB4 antibodies leads totumor regression.

FIG. 61 shows a genomic nucleotide sequence of human EphB4 (SEQ IDNO:6).

FIG. 62 shows a cDNA nucleotide sequence of human EphB4 (SEQ ID NO:7).

FIG. 63 shows a genomic nucleotide sequence of human Ephrin B2 (SEQ IDNO:8).

FIG. 64 shows a cDNA nucleotide sequence of human Ephrin B2 (SEQ IDNO:9).

FIG. 65 shows an amino acid sequence of human EphB4 (SEQ ID NO:10).

FIG. 66 shows an amino acid sequence of human Ephrin B2 (SEQ ID NO:11).

FIG. 67 shows a comparison of the EphrinB2 binding properties of theHSA-EphB4 fusion protein and other EphB4 polypeptides.

FIG. 68 shows a comparison between the in vivo stability of an EphB4-HSAfusion protein and an EphB4 polypeptide in mice.

FIG. 69 shows the EphrinB2 binding activity of soluble EphB4polypeptides pegylated under specific pH conditions.

FIG. 70 shows the chromatographic separation of PEG derivatives of EphB4protein on SP-Sepharose columns. Purity of the PEG-modified EphB4protein was analyzed by PAGE. The EphrinB2 binding of the pegylationreaction products is also shown.

FIG. 71 shows the purity, as determined by SDS-PAGE, ofchromatography-separated unpegylated, monopegylated and poly-pegylatedEphB4 fractions.

FIG. 72 shows the EphrinB2-binding activity of the chromatographyfractions from the EphB4 pegylation reaction.

FIG. 73 shows the retention of EphrinB2-binding activity of thechromatography fractions from the EphB4 pegylation reaction afterincubation in mouse serum at 37° C. for three days.

FIG. 74 shows the in vivo stability of unpegylated, monopegylated andpolypegylated EphB4 in mice over time.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The current invention is based in part on the discovery that signalingthrough the ephrin/ephrin receptor (ephrin/eph) pathway contributes totumorigenesis. Applicants detected expression of ephrin B2 and EphB4 intumor tissues and developed anti-tumor therapeutic agents for blockingsignaling through the ephrin/eph. In addition, the disclosure providespolypeptide therapeutic agents and methods for polypeptide-basedinhibition of the function of EphB4 and/or Ephrin B2. Accordingly, incertain aspects, the disclosure provides numerous polypeptide compounds(agents) that may be used to treat cancer as well as angiogenesisrelated disorders and unwanted angiogenesis related processes.Applicants have generated modified forms of EphrinB2 and EphB4polypeptides and have demonstrated that such modified forms havemarkedly improved pharmacokinetic properties. Accordingly, in certainaspects, the disclosure provides numerous polypeptide compounds (agents)that may be used to treat cancer as well as angiogenesis relateddisorders and unwanted angiogenesis related processes.

As used herein, the terms Ephrin and Eph are used to refer,respectively, to ligands and receptors. They can be from any of avariety of animals (e.g., mammals/non-mammals,vertebrates/non-vertebrates, including humans). The nomenclature in thisarea has changed rapidly and the terminology used herein is thatproposed as a result of work by the Eph Nomenclature Committee, whichcan be accessed, along with previously-used names at web sitehttp://www.eph-nomenclature.com.

The work described herein, particularly in the examples, refers toEphrin B2 and EphB4. However, the present invention contemplates anyephrin ligand and/or Eph receptor within their respective family, whichis expressed in a tumor. The ephrins (ligands) are of two structuraltypes, which can be further subdivided on the basis of sequencerelationships and, functionally, on the basis of the preferentialbinding they exhibit for two corresponding receptor subgroups.Structurally, there are two types of ephrins: those which aremembrane-anchored by a glycerophosphatidylinositol (GPI) linkage andthose anchored through a transmembrane domain. Conventionally, theligands are divided into the Ephrin-A subclass, which are GPI-linkedproteins which bind preferentially to EphA receptors, and the Ephrin-Bsubclass, which are transmembrane proteins which generally bindpreferentially to EphB receptors.

The Eph family receptors are a family of receptor protein-tyrosinekinases which are related to Eph, a receptor named for its expression inan erythropoietin-producing human hepatocellular carcinoma cell line.They are divided into two subgroups on the basis of the relatedness oftheir extracellular domain sequences and their ability to bindpreferentially to Ephrin-A proteins or Ephrin-B proteins. Receptorswhich interact preferentially with Ephrin-A proteins are EphA receptorsand those which interact preferentially with Ephrin-B proteins are EphBreceptors.

Eph receptors have an extracellular domain composed of theligand-binding globular domain, a cysteine rich region followed by apair of fibronectin type III repeats (e.g., see FIG. 16). Thecytoplasmic domain consists of a juxtamembrane region containing twoconserved tyrosine residues; a protein tyrosine kinase domain; a sterileα-motif (SAM) and a PDZ-domain binding motif. EphB4 is specific for themembrane-bound ligand Ephrin B2 (Sakano, S. et al 1996; Brambilla R. etal 1995). Ephrin B2 belongs to the class of Eph ligands that have atransmembrane domain and cytoplasmic region with five conserved tyrosineresidues and PDZ domain. Eph receptors are activated by binding ofclustered, membrane attached ephrins (Davis S et al, 1994), indicatingthat contact between cells expressing the receptors and cells expressingthe ligands is required for Eph activation.

Upon ligand binding, an Eph receptor dimerizes and autophosphorylate thejuxtamembrane tyrosine residues to acquire full activation (Kalo M S etal, 1999, Binns K S, 2000). In addition to forward signaling through theEph receptor, reverse signaling can occur through the ephrin Bs. Ephengagement of ephrins results in rapid phosphorylation of the conservedintracellular tyrosines (Bruckner K, 1997) and somewhat slowerrecruitment of PDZ binding proteins (Palmer A 2002). Recently, severalstudies have shown that high expression of Eph/ephrins may be associatedwith increased potentials for tumor growth, tumorigenicity, andmetastasis (Easty D J, 1999; Kiyokawa E, 1994; Tang X X, 1999; Vogt T,1998; Liu W, 2002; Stephenson S A, 2001; Steube K G 1999; Berclaz G,1996).

In certain embodiments, the present invention provides polypeptidetherapeutic agents that inhibit activity of Ephrin B2, EphB4, or both.As used herein, the term “polypeptide therapeutic agent” or “polypeptideagent” is a generic term which includes any polypeptide that blockssignaling through the Ephrin B2/EphB4 pathway. A preferred polypeptidetherapeutic agent of the invention is a soluble polypeptide of Ephrin B2or EphB4. Another preferred polypeptide therapeutic agent of theinvention is an antagonist antibody that binds to Ephrin B2 or EphB4.For example, such polypeptide therapeutic agent can inhibit function ofEphrin B2 or EphB4, inhibit the interaction between Ephrin B2 and EphB4,inhibit the phosphorylation of Ephrin B2 or EphB4, or inhibit any of thedownstream signaling events upon binding of Ephrin B2 to EphB4. Suchpolypeptides may include EphB4 or EphrinB2 that are modified so as toimprove serum half-life, such as by PEGylation or stable associationwith a serum albumin protein.

II. Soluble Polypeptides

In certain aspects, the invention relates to a soluble polypeptidecomprising an extracellular domain of an Ephrin B2 protein (referred toherein as an Ephrin B2 soluble polypeptide) or comprising anextracellular domain of an EphB4 protein (referred to herein as an EphB4soluble polypeptide). Preferably, the subject soluble polypeptide is amonomer and is capable of binding with high affinity to Ephrin B2 orEphB4. In a specific embodiment, the EphB4 soluble polypeptide of theinvention comprises a globular domain of an EphB4 protein. Specificexamples EphB4 soluble polypeptides are provided in FIGS. 1, 2, and 15.Specific examples of Ephrin B2 soluble polypeptides are provided inFIGS. 3 and 14.

As used herein, the subject soluble polypeptides include fragments,functional variants, and modified forms of EphB4 soluble polypeptide oran Ephrin B2 soluble polypeptide. These fragments, functional variants,and modified forms of the subject soluble polypeptides antagonizefunction of EphB4, Ephrin B2 or both.

In certain embodiments, isolated fragments of the subject solublepolypeptides can be obtained by screening polypeptides recombinantlyproduced from the corresponding fragment of the nucleic acid encoding anEphB4 or Ephrin B2 soluble polypeptides. In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function to inhibitfunction of EphB4 or Ephrin B2, for example, by testing the ability ofthe fragments to inhibit angiogenesis or tumor growth.

In certain embodiments, a functional variant of an EphB4 solublepolypeptide comprises an amino acid sequence that is at least 90%, 95%,97%, 99% or 100% identical to residues 1-197, 29-197, 1-312, 29-132,1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429,29-429, 1-526, 29-526, 1-537 and 29-537 of the amino acid sequencedefined by FIG. 65 (SEQ ID NO: 10). Such polypeptides may be used in aprocessed form, and accordingly, in certain embodiments, an EphB4soluble polypeptide comprises an amino acid sequence that is at least90%, 95%, 97%, 99% or 100% identical to residues 16-197, 16-312, 16-321,16-326, 16-412, 16-427, 16-429, 16-526 and 16-537 of the amino acidsequence defined by FIG. 65 (SEQ ID NO:10).

In other embodiments, a functional variant of an Ephrin B2 solublepolypeptide comprises a sequence at least 90%, 95%, 97%, 99% or 100%identical to residues 1-225 of the amino acid sequence defined by FIG.66 (SEQ ID NO: 11) or a processed form, such as one comprising asequence at least 90%, 95%, 97%, 99% or 100% identical to residues26-225 of the amino acid sequence defined by FIG. 66 (SEQ ID NO: 11).

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of the subject solublepolypeptide for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified soluble polypeptide areconsidered functional equivalents of the naturally-occurring EphB4 orEphrin B2 soluble polypeptide. Modified soluble polypeptides can beproduced, for instance, by amino acid substitution, deletion, oraddition. For instance, it is reasonable to expect, for example, that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the EphB4 or Ephrin B2 soluble polypeptides, aswell as truncation mutants, and is especially useful for identifyingfunctional variant sequences. The purpose of screening suchcombinatorial libraries may be to generate, for example, solublepolypeptide variants which can act as antagonists of EphB4, EphB2, orboth. Combinatorially-derived variants can be generated which have aselective potency relative to a naturally occurring soluble polypeptide.Such variant proteins, when expressed from recombinant DNA constructs,can be used in gene therapy protocols. Likewise, mutagenesis can giverise to variants which have intracellular half-lives dramaticallydifferent than the corresponding wild-type soluble polypeptide. Forexample, the altered protein can be rendered either more stable or lessstable to proteolytic degradation or other cellular process which resultin destruction of, or otherwise inactivation of the protein of interest(e.g., a soluble polypeptide). Such variants, and the genes which encodethem, can be utilized to alter the subject soluble polypeptide levels bymodulating their half-life. For instance, a short half-life can giverise to more transient biological effects and, when part of an inducibleexpression system, can allow tighter control of recombinant solublepolypeptide levels within the cell. As above, such proteins, andparticularly their recombinant nucleic acid constructs, can be used ingene therapy protocols.

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriategene for expression. The purpose of a degenerate set of genes is toprovide, in one mixture, all of the sequences encoding the desired setof potential soluble polypeptide sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic AcidRes. 11:477). Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al., (1990)Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433;Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNASUSA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, soluble polypeptide variants (e.g.,the antagonist forms) can be generated and isolated from a library byscreening using, for example, alanine scanning mutagenesis and the like(Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J.Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118;Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al.,(1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), bylinker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660;Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al.,(1982) Science 232:316); by saturation mutagenesis (Meyers et al.,(1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) MethodCell Mol Biol 1:11-19); or by random mutagenesis, including chemicalmutagenesis, etc. (Miller et al., (1992) A Short Course in BacterialGenetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al.,(1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis,particularly in a combinatorial setting, is an attractive method foridentifying truncated (bioactive) forms of the subject solublepolypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the subject soluble polypeptides. The mostwidely used techniques for screening large gene libraries typicallycomprises cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, the subject soluble polypeptides of theinvention include a small molecule such as a peptide and apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the EphB4 or Ephrin B2 solublepolypeptides.

In certain embodiments, the soluble polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modification include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. Effects of suchnon-amino acid elements on the functionality of a soluble polypeptidemay be tested for its antagonizing role in EphB4 or Ephrin B2 function,e.g, it inhibitory effect on angiogenesis or on tumor growth.

In one specific embodiment of the present invention, modified forms ofthe subject soluble polypeptides comprise linking the subject solublepolypeptides to nonproteinaceous polymers. In one specific embodiment,the polymer is polyethylene glycol (“PEG”), polypropylene glycol, orpolyoxyalkylenes, in the manner as set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Examples of themodified polypeptide of the invention include PEGylated soluble EphrinB2 and PEGylated soluble EphB4.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula:

X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH (1), where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. In one embodiment, the PEG ofthe invention terminates on one end with hydroxy or methoxy, i.e., X isH or CH₃ (“methoxy PEG”). A PEG can contain further chemical groupswhich are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that an EphB4 containing a PEG molecule is alsoknown as a conjugated protein, whereas the protein lacking an attachedPEG molecule can be referred to as unconjugated.

Any molecular mass for a PEG can be used as practically desired, e.g.,from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to Eph4 or EphrinB2 soluble peptides. The number ofrepeating units “n” in the PEG is approximated for the molecular massdescribed in Daltons. It is preferred that the combined molecular massof PEG on an activated linker is suitable for pharmaceutical use. Thus,in one embodiment, the molecular mass of the PEG molecules does notexceed 100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12,000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment of the invention, an EphB4 polypeptide iscovalently linked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of EphB4; R being lower alkyl; x being 2 or 3; m being from about450 to about 950; and n and m being chosen so that the molecular weightof the conjugate minus the EphB4 protein is from about 10 to 40 kDa. Inone embodiment, an EphB4 ε-amino group of a lysine is the available(free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR(II), wherein P is the group of anEphB4 protein as described herein, (i.e. without the amino group oramino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the EphB4 protein is from about 10 to about 40 kDa. Asused herein, the given ranges of “m” have an orientational meaning. Theranges of “m” are determined in any case, and exactly, by the molecularweight of the PEG group.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated EphB4 will be used therapeutically, thedesired dosage, circulation time, resistance to proteolysis,immunogenicity, and other considerations. For a discussion of PEG andits use to enhance the properties of proteins, see N. V. Kate, AdvancedDrug Delivery Reviews 10: 91-114 (1993).

In one embodiment of the invention, PEG molecules may be activated toreact with amino groups on EphB4, such as with lysines (Bencham C. O. etal., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl.Biochem., 11, 141 (1985); Zalipsky, S. et al., Polymeric Drugs and DrugDelivery Systems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky,S. et al., Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al.,Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

In one specific embodiment, carbonate esters of PEG are used to form thePEG-EphB4 conjugates. N,N′-disuccinimidylcarbonate (DSC) may be used inthe reaction with PEG to form active mixed PEG-succinimidyl carbonatethat may be subsequently reacted with a nucleophilic group of a linkeror an amino group of EphB4 (see U.S. Pat. No. 5,281,698 and U.S. Pat.No. 5,932,462). In a similar type of reaction,1,1′-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may bereacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixedcarbonate (U.S. Pat. No. 5,382,657), respectively.

In one embodiment, additional sites for PEGylation are introduced bysite-directed mutagenesis by introducing one or more lysine residues.For instance, one or more arginine residues may be mutated to a lysineresidue. In another embodiment, additional PEGylation sites arechemically introduced by modifying amino acids on EphB4. In one specificembodiment, carboxyl groups in EphB4 are conjugated with diaminobutane,resulting in carboxyl amidation (see Li et al., Anal Biochem. 2004;330(2):264-71). This reaction may be catalyzed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water-solublecarbodiimide. The resulting amides can then conjugated to PEG.

PEGylation of EphB4 can be performed according to the methods of thestate of the art, for example by reaction of EphB4 withelectrophilically active PEGs (supplier: Shearwater Corp., USA,www.shearwatercorp.com). Preferred PEG reagents of the present inventionare, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA), butanoates(PEG-SBA), PEG-succinimidyl propionate or branched N-hydroxysuccinimidessuch as mPEG2-NHS (Monfardini, C., et al., Bioconjugate Chem. 6 (1995)62-69). Such methods may used to PEGylated at an ε-amino group of anEphB4 lysine or the N-terminal amino group of EphB4.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson EphB4 (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45(1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson etal., Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat.Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEG speciesthat may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on EphB4, the cysteine residues are native to Eph4, whereas inother embodiments, one or more cysteine residues are engineered intoEphB4. Mutations may be introduced into an EphB4 coding sequence togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein. Alternatively, surface residuesmay be predicted by comparing the amino acid sequences of EphB4 anEphB2, given that the crystal structure of EphB2 has been solved (seeHimanen et al., Nature. (2001) 20-27; 414(6866):933-8) and thus thesurface-exposed residues identified. In one embodiment, cysteineresidues are introduced into EphB4 at or near the N- and/or C-terminus,or within loop regions. Loop regions may be identified by comparing theEphB4 sequence to that of EphB2.

In some embodiments, the pegylated EphB4 comprises a PEG moleculecovalently attached to the alpha amino group of the N-terminal aminoacid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) WET, 297,1059, and U.S. Pat. No. 5,824,784. Theuse of a PEG-aldehyde for the reductive amination of a protein utilizingother available nucleophilic amino groups is described in U.S. Pat. No.4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and inChamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated EphB4 comprises one or more PEGmolecules covalently attached to a linker, which in turn is attached tothe alpha amino group of the amino acid residue at the N-terminus ofEphB4. Such an approach is disclosed in U.S. Patent Publication No.2002/0044921 and in WO94/01451.

In one embodiment, EphB4 is pegylated at the C-terminus. In a specificembodiment, a protein is pegylated at the C-terminus by the introductionof C-terminal azido-methionine and the subsequent conjugation of amethyl-PEG-triarylphosphine compound via the Staudinger reaction. ThisC-terminal conjugation method is described in Cazalis et al., C-TerminalSite-Specific PEGylation of a Truncated Thrombomodulin Mutant withRetention of Full Bioactivity, Bioconjug Chem. 2004; 15(5):1005-1009.

Monopegylation of EphB4 can also be produced according to the generalmethods described in WO 94/01451. WO 94/01451 describes a method forpreparing a recombinant polypeptide with a modified terminal amino acidalpha-carbon reactive group. The steps of the method involve forming therecombinant polypeptide and protecting it with one or more biologicallyadded protecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino acid alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with a chemical modifying agent. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of EphB4 (or EphrinB2) to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to EphB4. Inone embodiment, the pH of a buffer used is from about 7.0 to 9.0. Inanother embodiment, the pH is in a slightly basic range, e.g., fromabout 7.5 to 8.5. Buffers having a pKa close to neutral pH range may beused, e.g., phosphate buffer.

In one embodiment, the temperature range for preparing a mono-PEG-EphB4is from about 4° C. to 40° C., or from about 18° C. to 25° C. In anotherembodiment, the temperature is room temperature.

The pegylation reaction can proceed from 3 to 48 hours, or from 10 to 24hours. The reaction can be monitored using SE-HPLC to distinguish EphB4,mono-PEG-EphB4 and poly-PEG-EphB4. It is noted that mono-PEG-EphB4 formsbefore di-PEG-EphB4. When the mono-PEG-EphB4 concentration reaches aplateau, the reaction can be terminated by adding a quenching agent toreact with unreacted PEG. In some embodiments, the quenching agent is afree amino acid, such as glycine, cysteine or lysine.

Conventional separation and purification techniques known in the art canbe used to purify pegylated EphB4 or EphrinB2 products, such as sizeexclusion (e.g. gel filtration) and ion exchange chromatography.Products may also be separated using SDS-PAGE. Products that may beseparated include mono-, di-, tri- poly- and un-pegylated EphB4, as wellas free PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, pegylated EphB4 proteins of the invention containone, two or more PEG moieties. In one embodiment, the PEG moiety(ies)are bound to an amino acid residue which is on the surface of theprotein and/or away from the surface that contacts EphrinB2. In oneembodiment, the combined or total molecular mass of PEG in PEG-EphB4 isfrom about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to36,000 Da. In a one embodiment, the PEG in pegylated EphB4 is asubstantially linear, straight-chain PEG.

In one embodiment of the invention, the PEG in pegylated EphB4 orEphrinB2 is not hydrolyzed from the pegylated amino acid residue using ahydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8 to 16hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-EphB4, morepreferably at least 90%, and most preferably at least 95%.

In another embodiment, the pegylated EphB4 proteins of the inventionwill preferably retain at least 25%, 50%, 60%, 70% least 80%, 85%, 90%,95% or 100% of the biological activity associated with the unmodifiedprotein. In one embodiment, biological activity refers to its ability tobind to EphrinB2. In one specific embodiment, the pegylated EphB4protein shows an increase in binding to EphrinB2 relative to unpegylatedEphB4.

In a preferred embodiment, the PEG-EphB4 has a half-life (t_(1/2)) whichis enhanced relative to the half-life of the unmodified protein.Preferably, the half-life of PEG-EphB4 is enhanced by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%,300%, 400% or 500%, or even by 1000% relative to the half-life of theunmodified EphB4 protein. In some embodiments, the protein half-life isdetermined in vitro, such as in a buffered saline solution or in serum.In other embodiments, the protein half-life is an in vivo half life,such as the half-life of the protein in the serum or other bodily fluidof an animal.

In certain aspects, functional variants or modified forms of the subjectsoluble polypeptides include fusion proteins having at least a portionof the soluble polypeptide and one or more fusion domains. Well knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin,protein A, protein G, and an immunoglobulin heavy chain constant region(Fc), maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Another fusion domain well known inthe art is green fluorescent protein (GFP). Fusion domains also include“epitope tags,” which are usually short peptide sequences for which aspecific antibody is available. Well known epitope tags for whichspecific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation.

In certain embodiments, the soluble polypeptides of the presentinvention contain one pr more modifications that are capable ofstabilizing the soluble polypeptides. For example, such modificationsenhance the in vitro half life of the soluble polypeptides, enhancecirculatory half life of the soluble polypeptides or reducingproteolytic degradation of the soluble polypeptides.

In a further embodiment, a soluble polypeptide of the present inventionis fused to a cytotoxic agent. In this method, the fusion acts to targetthe cytotoxic agent to a specific tissue or cell (e.g., a tumor tissueor cell), resulting in a reduction in the number of afflicted cells.Such an approach can thereby reduce symptoms associated with cancer andangiogenesis-associated disorders. Cytotoxic agents include, but are notlimited to, diphtheria A chain, exotoxin A chain, ricin A chain, abrin Achain, curcin, crotin, phenomycin, enomycin and the like, as well asradiochemicals.

In certain embodiments, the soluble polypeptides of the presentinvention may be fused to other therapeutic proteins or to otherproteins such as Fc or serum albumin for pharmacokinetic purposes. Seefor example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which areincorporated by reference. In some embodiments, soluble peptides of thepresent invention are fused to Fc variants. In a specific embodiment,the soluble polypeptide is fused to an Fc variant which does nothomodimerize, such as one lacking the cysteine residues which formcysteine bonds with other Fc chains.

In some embodiments, the modified proteins of the invention comprisefusion proteins with an Fc region of an immunoglobulin. As is known,each immunoglobulin heavy chain constant region comprises four or fivedomains. The domains are named sequentially as follows:CH1-hinge-CH2-CH3(-CH₄). The DNA sequences of the heavy chain domainshave cross-homology among the immunoglobulin classes, e.g., the CH2domain of IgG is homologous to the CH2 domain of IgA and IgD, and to theCH3 domain of IgM and IgE. As used herein, the term, “immunoglobulin Fcregion” is understood to mean the carboxyl-terminal portion of animmunoglobulin chain constant region, preferably an immunoglobulin heavychain constant region, or a portion thereof. For example, animmunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, anda CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and aCH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of twoor more domains and an immunoglobulin hinge region. In a preferredembodiment the immunoglobulin Fc region comprises at least animmunoglobulin hinge region a CH2 domain and a CH3 domain, andpreferably lacks the CH1 domain.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). The nucleotide and amino acid sequences of human Fc .gamma.-1 areset forth in SEQ ID NOS: 5 and 6. The nucleotide and amino acidsequences of murine Fcγ-2a are set forth in SEQ ID NOS: 7 and 8. Otherclasses of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM(Igμ), may be used. The choice of appropriate immunoglobulin heavy chainconstant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and5,726,044. The choice of particular immunoglobulin heavy chain constantregion sequences from certain immunoglobulin classes and subclasses toachieve a particular result is considered to be within the level ofskill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH₃ domain of Fc γor the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the invention. One example would be tointroduce amino acid substitutions in the upper CH2 region to create aFc variant with reduced affinity for Fc receptors (Cole et al. (1997) J.IMMUNOL. 159:3613). One of ordinary skill in the art can prepare suchconstructs using well known molecular biology techniques.

In a specific embodiment of the present invention, the modified forms ofthe subject soluble polypeptides are fusion proteins having at least aportion of the soluble polypeptide (e.g., an ectodomain of Ephrin B2 orEphB4) and a stabilizing domain such as albumin. As used herein,“albumin” refers collectively to albumin protein or amino acid sequence,or an albumin fragment or variant, having one or more functionalactivities (e.g., biological activities) of albumin. In particular,“albumin” refers to human albumin or fragments thereof (see EP 201 239,EP 322 094 WO 97/24445, WO95/23857) especially the mature form of humanalbumin, or albumin from other vertebrates or fragments thereof, oranalogs or variants of these molecules or fragments thereof.

The present invention describes that such fusion proteins are morestable relative to the corresponding wildtype soluble protein. Forexample, the subject soluble polypeptide (e.g., an ectodomain of EphrinB2 or EphB4) can be fused with human serum albumin (HSA), bovine serumalbumin (BSA), or any fragment of an albumin protein which hasstabilization activity. Such stabilizing domains include human serumalbumin (HSA) and bovine serum albumin (BSA).

In particular, the albumin fusion proteins of the invention may includenaturally occurring polymorphic variants of human albumin and fragmentsof human albumin (See WO95/23857), for example those fragments disclosedin EP 322 094 (namely HA (Pn), where n is 369 to 419). The albumin maybe derived from any vertebrate, especially any mammal, for examplehuman, cow, sheep, or pig. Non-mammalian albumins include, but are notlimited to, hen and salmon. The albumin portion of the albumin fusionprotein may be from a different animal than the EphB4.

In some embodiments, the albumin protein portion of an albumin fusionprotein corresponds to a fragment of serum albumin. Fragments of serumalbumin polypeptides include polypeptides having one or more residuesdeleted from the amino terminus or from the C-terminus. Generallyspeaking, an HA fragment or variant will be at least 100 amino acidslong, preferably at least 150 amino acids long. The HA variant mayconsist of or alternatively comprise at least one whole domain of HA.Domains, with reference to SEQ ID NO:18 in U.S. Patent Publication No.2004/0171123, are as follows: domains 1 (amino acids 1-194), 2 (aminoacids 195-387), 3 (amino acids 388-585), 1+2 (1-387), 2+3 (195-585) or1+3 (amino acids 1-194+amino acids 388-585). Each domain is itself madeup of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387,388-491 and 512-585, with flexible inter-subdomain linker regionscomprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492 toAla511.

In one embodiment, the EphB4-HSA fusion has one EphB4 solublepolypeptide linked to one HSA molecule, but other conformations arewithin the invention. For example, EphB4-HSA fusion proteins can haveany of the following formula: R₁-L-R₂; R₂-L-R₁; R₁-L-R₂-L-R₁; orR₂-L-R1-L-R₂; R₁-R₂; R₂-R₁; R₁-R₂-R₁; or R₂-R₁-R₂; wherein R₁ is asoluble EphB4 sequence, R₂ is HSA, and L is a peptide linker sequence.

In a specific embodiment, the EphB4 and HSA domains are linked to eachother, preferably via a linker sequence, which separates the EphB4 andHSA domains by a distance sufficient to ensure that each domain properlyfolds into its secondary and tertiary structures. Preferred linkersequences (1) should adopt a flexible extended conformation, (2) shouldnot exhibit a propensity for developing an ordered secondary structurewhich could interact with the functional EphB4 and HSA domains, and (3)should have minimal hydrophobic or charged character, which couldpromote interaction with the functional protein domains. Typical surfaceamino acids in flexible protein regions include Gly, Asn and Ser.Permutations of amino acid sequences containing Gly, Asn and Ser wouldbe expected to satisfy the above criteria for a linker sequence. Othernear neutral amino acids, such as Thr and Ala, can also be used in thelinker sequence.

In a specific embodiment, a linker sequence length of about 20 aminoacids can be used to provide a suitable separation of functional proteindomains, although longer or shorter linker sequences may also be used.The length of the linker sequence separating EphB4 and HSA can be from 5to 500 amino acids in length, or more preferably from 5 to 100 aminoacids in length. Preferably, the linker sequence is from about 5-30amino acids in length. In preferred embodiments, the linker sequence isfrom about 5 to about 20 amino acids, and is advantageously from about10 to about 20 amino acids. Amino acid sequences useful as linkers ofEphB4 and HSA include, but are not limited to, (SerGly₄)y wherein y isgreater than or equal to 8, or Gly₄SerGly₅Ser. A preferred linkersequence has the formula (SerGly₄)₄. Another preferred linker has thesequence ((Ser-Ser-Ser-Ser-Gly)3-Ser-Pro).

In one embodiment, the polypeptides of the present invention and HSAproteins are directly fused without a linker sequence. In preferredembodiments, the C-terminus of a soluble EphB4 polypeptide can bedirectly fused to the N-terminus of HSA or the C-terminus of HSA can bedirectly fused to the N-terminus of soluble EphB4.

In some embodiments, the immunogenicity of the fusion junction betweenHSA and EphB4 may be reduced the by identifying a candidate T-cellepitope within a junction region spanning a fusion protein and changingan amino acid within the junction region as described in U.S. PatentPublication No. 2003/0166877.

In certain embodiments, soluble polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such soluble polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the soluble polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems as is well known in the art (also see below).

III. Nucleic Acids Encoding Soluble Polypeptides

In certain aspects, the invention relates to isolated and/or recombinantnucleic acids encoding an EphB4 or Ephrin B2 soluble polypeptide. Thesubject nucleic acids may be single-stranded or double-stranded, DNA orRNA molecules. These nucleic acids are useful as therapeutic agents. Forexample, these nucleic acids are useful in making recombinant solublepolypeptides which are administered to a cell or an individual astherapeutics. Alternative, these nucleic acids can be directlyadministered to a cell or an individual as therapeutics such as in genetherapy.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to a region of the nucleotide sequence depicted inSEQ ID Nos. 6-9. One of ordinary skill in the art will appreciate thatnucleic acid sequences complementary to the subject nucleic acids, andvariants of the subject nucleic acids are also within the scope of thisinvention. In further embodiments, the nucleic acid sequences of theinvention can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence depicted in SEQ ID Nos. 6-9, or complementsequences thereof. As discussed above, one of ordinary skill in the artwill understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. One of ordinary skill in theart will understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the subject nucleic acids dueto degeneracy in the genetic code are also within the scope of theinvention. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate for a host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspect of the invention, the subject nucleic acid is providedin an expression vector comprising a nucleotide sequence encoding anEphB4 or Ephrin B2 soluble polypeptide and operably linked to at leastone regulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the soluble polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a soluble polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, the lac system, the trp system, the TAC or TRC system,T7 promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject soluble polypeptide. The host cell may be any prokaryotic oreukaryotic cell. For example, a soluble polypeptide of the invention maybe expressed in bacterial cells such as E. coli, insect cells (e.g.,using a baculovirus expression system), yeast, or mammalian cells. Othersuitable host cells are known to those skilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject soluble polypeptides. For example, a host celltransfected with an expression vector encoding an EphB4 solublepolypeptide can be cultured under appropriate conditions to allowexpression of the EphB4 soluble polypeptide to occur. The EphB4 solublepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the soluble polypeptides. Alternatively, the solublepolypeptides may be retained cytoplasmically or in a membrane fractionand the cells harvested, lysed and the protein isolated. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The soluble polypeptides can beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for particular epitopes of the soluble polypeptides. In apreferred embodiment, the soluble polypeptide is a fusion proteincontaining a domain which facilitates its purification.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant soluble polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Examplesof other viral (including retroviral) expression systems can be foundbelow in the description of gene therapy delivery systems. The variousmethods employed in the preparation of the plasmids and transformationof host organisms are well known in the art. For other suitableexpression systems for both prokaryotic and eukaryotic cells, as well asgeneral recombinant procedures, see Molecular Cloning A LaboratoryManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, itmay be desirable to express the recombinant SLC5A8 polypeptide by theuse of a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUW1), andpBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

IV. Drug Screening Assays

There are numerous approaches to screening for polypeptide therapeuticagents as antagonists of EphB4, Ephrin B2 or both. For example,high-throughput screening of compounds or molecules can be carried outto identify agents or drugs which inhibit angiogenesis or inhibit tumorgrowth. Test agents can be any chemical (element, molecule, compound,drug), made synthetically, made by recombinant techniques or isolatedfrom a natural source. For example, test agents can be peptides,polypeptides, peptoids, sugars, hormones, or nucleic acid molecules. Inaddition, test agents can be small molecules or molecules of greatercomplexity made by combinatorial chemistry, for example, and compiledinto libraries. These libraries can comprise, for example, alcohols,alkyl halides, amines, amides, esters, aldehydes, ethers and otherclasses of organic compounds. Test agents can also be natural orgenetically engineered products isolated from lysates or growth media ofcells—bacterial, animal or plant—or can be the cell lysates or growthmedia themselves. Presentation of test compounds to the test system canbe in either an isolated form or as mixtures of compounds, especially ininitial screening steps.

For example, an assay can be carried out to screen for compounds thatspecifically inhibit binding of Ephrin B2 (ligand) to EphB4 (receptor),or vice-versa, e.g., by inhibition of binding of labeled ligand- orreceptor-Fc fusion proteins to immortalized cells. Compounds identifiedthrough this screening can then be tested in animals to assess theiranti-angiogenesis or anti-tumor activity in vivo.

In one embodiment of an assay to identify a substance that interfereswith interaction of two cell surface molecules (e.g., Ephrin B2 andEphB4), samples of cells expressing one type of cell surface molecule(e.g., EphB4) are contacted with either labeled ligand (e.g., Ephrin B2,or a soluble portion thereof, or a fusion protein such as a fusion ofthe extracellular domain and the Fc domain of IgG) or labeled ligandplus a test compound (or group of test compounds). The amount of labeledligand which has bound to the cells is determined. A lesser amount oflabel (where the label can be, for example, a radioactive isotope, afluorescent or colorimetric label) in the sample contacted with the testcompound(s) is an indication that the test compound(s) interferes withbinding. The reciprocal assay using cells expressing a ligand (e.g., anEphrin B2 ligand or a soluble form thereof) can be used to test for asubstance that interferes with the binding of an Eph receptor or solubleportion thereof.

An assay to identify a substance which interferes with interactionbetween an Eph receptor and an ephrin can be performed with thecomponent (e.g., cells, purified protein, including fusion proteins andportions having binding activity) which is not to be in competition witha test compound, linked to a solid support. The solid support can be anysuitable solid phase or matrix, such as a bead, the wall of a plate orother suitable surface (e.g., a well of a microtiter plate), column poreglass (CPG) or a pin that can be submerged into a solution, such as in awell. Linkage of cells or purified protein to the solid support can beeither direct or through one or more linker molecules.

In one embodiment, an isolated or purified protein (e.g., an Ephreceptor or an ephrin) can be immobilized on a suitable affinity matrixby standard techniques, such as chemical cross-linking, or via anantibody raised against the isolated or purified protein, and bound to asolid support. The matrix can be packed in a column or other suitablecontainer and is contacted with one or more compounds (e.g., a mixture)to be tested under conditions suitable for binding of the compound tothe protein. For example, a solution containing compounds can be made toflow through the matrix. The matrix can be washed with a suitable washbuffer to remove unbound compounds and non-specifically bound compounds.Compounds which remain bound can be released by a suitable elutionbuffer. For example, a change in the ionic strength or pH of the elutionbuffer can lead to a release of compounds. Alternatively, the elutionbuffer can comprise a release component or components designed todisrupt binding of compounds (e.g., one or more ligands or receptors, asappropriate, or analogs thereof which can disrupt binding orcompetitively inhibit binding of test compound to the protein).

Fusion proteins comprising all, or a portion of, a protein (e.g., an Ephreceptor or an ephrin) linked to a second moiety not occurring in thatprotein as found in nature can be prepared for use in another embodimentof the method. Suitable fusion proteins for this purpose include thosein which the second moiety comprises an affinity ligand (e.g., anenzyme, antigen, epitope). The fusion proteins can be produced byinserting the protein (e.g., an Eph receptor or an ephrin) or a portionthereof into a suitable expression vector which encodes an affinityligand. The expression vector can be introduced into a suitable hostcell for expression. Host cells are disrupted and the cell material,containing fusion protein, can be bound to a suitable affinity matrix bycontacting the cell material with an affinity matrix under conditionssufficient for binding of the affinity ligand portion of the fusionprotein to the affinity matrix.

In one aspect of this embodiment, a fusion protein can be immobilized ona suitable affinity matrix under conditions sufficient to bind theaffinity ligand portion of the fusion protein to the matrix, and iscontacted with one or more compounds (e.g., a mixture) to be tested,under conditions suitable for binding of compounds to the receptor orligand protein portion of the bound fusion protein. Next, the affinitymatrix with bound fusion protein can be washed with a suitable washbuffer to remove unbound compounds and non-specifically bound compoundswithout significantly disrupting binding of specifically boundcompounds. Compounds which remain bound can be released by contactingthe affinity matrix having fusion protein bound thereto with a suitableelution buffer (a compound elution buffer). In this aspect, compoundelution buffer can be formulated to permit retention of the fusionprotein by the affinity matrix, but can be formulated to interfere withbinding of the compound(s) tested to the receptor or ligand proteinportion of the fusion protein. For example, a change in the ionicstrength or pH of the elution buffer can lead to release of compounds,or the elution buffer can comprise a release component or componentsdesigned to disrupt binding of compounds to the receptor or ligandprotein portion of the fusion protein (e.g., one or more ligands orreceptors or analogs thereof which can disrupt binding of compounds tothe receptor or ligand protein portion of the fusion protein).Immobilization can be performed prior to, simultaneous with, or aftercontacting the fusion protein with compound, as appropriate. Variouspermutations of the method are possible, depending upon factors such asthe compounds tested, the affinity matrix selected, and elution bufferformulation. For example, after the wash step, fusion protein withcompound bound thereto can be eluted from the affinity matrix with asuitable elution buffer (a matrix elution buffer). Where the fusionprotein comprises a cleavable linker, such as a thrombin cleavage site,cleavage from the affinity ligand can release a portion of the fusionwith compound bound thereto. Bound compound can then be released fromthe fusion protein or its cleavage product by an appropriate method,such as extraction.

V. Methods of Treatment

In certain embodiments, the present invention provides methods ofinhibiting angiogenesis and methods of treating angiogenesis-associateddiseases. In other embodiments, the present invention provides methodsof inhibiting or reducing tumor growth and methods of treating anindividual suffering from cancer. These methods involve administering tothe individual a therapeutically effective amount of one or morepolypeptide therapeutic agents as described above. These methods areparticularly aimed at therapeutic and prophylactic treatments ofanimals, and more particularly, humans.

As described herein, angiogenesis-associated diseases include, but arenot limited to, angiogenesis-dependent cancer, including, for example,solid tumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; inflammatorydisorders such as immune and non-immune inflammation; chronic articularrheumatism and psoriasis; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;telangiectasia psoriasis scleroderma, pyogenic granuloma, rubeosis,arthritis, diabetic neovascularization, vasculogenesis, hematopoiesis.

It is understood that methods and compositions of the invention are alsouseful for treating any angiogenesis-independent cancers (tumors). Asused herein, the term “angiogenesis-independent cancer” refers to acancer (tumor) where there is no or little neovascularization in thetumor tissue.

In particular, polypeptide therapeutic agents of the present inventionare useful for treating or preventing a cancer (tumor), including, butnot limited to, colon carcinoma, breast cancer, mesothelioma, prostatecancer, bladder cancer, squamous cell carcinoma of the head and neck(HNSCC), Kaposi sarcoma, and leukemia.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject methods of the invention can be usedalone. Alternatively, the subject methods may be used in combinationwith other conventional anti-cancer therapeutic approaches directed totreatment or prevention of proliferative disorders (e.g., tumor). Forexample, such methods can be used in prophylactic cancer prevention,prevention of cancer recurrence and metastases after surgery, and as anadjuvant of other conventional cancer therapy. The present inventionrecognizes that the effectiveness of conventional cancer therapies(e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, andsurgery) can be enhanced through the use of a subject polypeptidetherapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent isshown to enhance the therapeutic effect of the anti-neoplastic agent orovercome cellular resistance to such anti-neoplastic agent. This allowsdecrease of dosage of an anti-neoplastic agent, thereby reducing theundesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by theirmechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes—dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (vascular endothelial growth factor (VEGF) inhibitors,fibroblast growth factor (FGF) inhibitors); angiotensin receptorblocker; nitric oxide donors; anti-sense oligonucleotides; antibodies(trastuzumab); cell cycle inhibitors and differentiation inducers(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,hydrocortisone, methylpednisolone, prednisone, and prenisolone); growthfactor signal transduction kinase inhibitors; mitochondrial dysfunctioninducers and caspase activators; and chromatin disruptors.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory anti-angiogenesis therapy include: (1) inhibitors of releaseof “angiogenic molecules,” such as bFGF (basic fibroblast growthfactor); (2) neutralizers of angiogenic molecules, such as an anti-βbFGFantibodies; and (3) inhibitors of endothelial cell response toangiogenic stimuli, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like. For additional proposed inhibitors ofangiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118(1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab.Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946,5,192,744, 5,202,352, and 6,573,256. In addition, there are a widevariety of compounds that can be used to inhibit angiogenesis, forexample, peptides or agents that block the VEGF-mediated angiogenesispathway, endostatin protein or derivatives, lysine binding fragments ofangiostatin, melanin or melanin-promoting compounds, plasminogenfragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits,antagonists of vitronectin α_(v)β₃, peptides derived from Saposin B,antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810,6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103,6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents of the invention may be continuedwhile the other therapy is being administered and/or thereafter.Administration of the polypeptide therapeutic agents may be made in asingle dose, or in multiple doses. In some instances; administration ofthe polypeptide therapeutic agents is commenced at least several daysprior to the conventional therapy, while in other instances,administration is begun either immediately before or at the time of theadministration of the conventional therapy.

VI. Methods of Administration and Pharmaceutical Compositions

In certain embodiments, the subject polypeptide therapeutic agents(e.g., soluble polypeptides or antibodies) of the present invention areformulated with a pharmaceutically acceptable carrier. Such therapeuticagents can be administered alone or as a component of a pharmaceuticalformulation (composition). The compounds may be formulated foradministration in any convenient way for use in human or veterinarymedicine. Wetting agents, emulsifiers and lubricants, such as sodiumlauryl sulfate and magnesium stearate, as well as coloring agents,release agents, coating agents, sweetening, flavoring and perfumingagents, preservatives and antioxidants can also be present in thecompositions.

Formulations of the subject polypeptide therapeutic agents include thosesuitable for oral/nasal, topical, parenteral, rectal, and/orintravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage, formwill vary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations orcompositions include combining another type of anti-tumor oranti-angiogenesis therapeutic agent and a carrier and, optionally, oneor more accessory ingredients. In general, the formulations can beprepared with a liquid carrier, or a finely divided solid carrier, orboth, and then, if necessary, shaping the product.

Formulations for oral administration may be in the form of capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a subject polypeptide therapeutic agent as anactive ingredient.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more polypeptidetherapeutic agents of the present invention may be mixed with one ormore pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In particular, methods of the invention can be administered topically,either to skin or to mucosal membranes such as those on the cervix andvagina. This offers the greatest opportunity for direct delivery totumor with the lowest chance of inducing side effects. The topicalformulations may further include one or more of the wide variety ofagents known to be effective as skin or stratum corneum penetrationenhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone,dimethylacetamide, dimethylformamide, propylene glycol, methyl orisopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents mayfurther be included to make the formulation cosmetically acceptable.Examples of these are fats, waxes, oils, dyes, fragrances,preservatives, stabilizers, and surface active agents. Keratolyticagents such as those known in the art may also be included. Examples aresalicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The subject polypeptide therapeutic agents maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required. The ointments, pastes, creams and gels may contain, inaddition to a subject polypeptide agent, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject polypeptidetherapeutic agent, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates, and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more polypeptide therapeutic agents in combination withone or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofone or more polypeptide therapeutic agents in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of drug topolymer, and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Formulations for intravaginal or rectally administration may bepresented as a suppository, which may be prepared by mixing one or morecompounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound.

In other embodiments, the polypeptide therapeutic agents of the instantinvention can be expressed within cells from eukaryotic promoters. Forexample, a soluble polypeptide of EphB4 or Ephrin B2 can be expressed ineukaryotic cells from an appropriate vector. The vectors are preferablyDNA plasmids or viral vectors. Viral vectors can be constructed basedon, but not limited to, adeno-associated virus, retrovirus, adenovirus,or alphavirus. Preferably, the vectors stably introduced in and persistin target cells. Alternatively, viral vectors can be used that providefor transient expression. Such vectors can be repeatedly administered asnecessary. Delivery of vectors encoding the subject polypeptidetherapeutic agent can be systemic, such as by intravenous orintramuscular administration, by administration to target cellsex-planted from the patient followed by reintroduction into the patient,or by any other means that would allow for introduction into the desiredtarget cell (for a review see Couture et al., 1996, TIG., 12, 510).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Soluble Derivatives of the Extracellular Domains of HumanEphrin B2 and EphB4 Proteins

Soluble derivatives of the extracellular domains of human Ephrin B2 andEphB4 proteins represent either truncated full-length predictedextracellular domains of Ephrin B2 (B4ECv3, B2EC) or translationalfusions of the domains with constant region of human immunoglobulins(IgG1 Fc fragment), such as B2EC-FC, B4ECv2-FC and B4ECv3-FC.Representative human Ephrin B2 constructs and human EphB4 constructs areshown FIGS. 14 and 15.

The cDNA fragments encoding these recombinant proteins were subclonedinto mammalian expression vectors, expressed in transiently or stablytransfected mammalian cell lines and purified to homogeneity asdescribed in detail in Materials and Methods section (see below).Predicted amino acid sequences of the proteins are shown in FIGS. 1-5.High purity of the isolated proteins and their recognition by thecorresponding anti-Ephrin B2 and anti-EphB4 monoclonal or polyclonalantibodies were confirmed. The recombinant proteins exhibit the expectedhigh-affinity binding, binding competition and specificity propertieswith their corresponding binding partners as corroborated by thebiochemical assays (see e.g., FIGS. 6-8).

Such soluble derivative proteins human Ephrin B2 and EphB4 exhibitpotent biological activity in several cell-based assays and in vivoassays which measure angiogenesis or anti-cancer activities, and aretherefore perspective drug candidates for anti-angiogenic andanti-cancer therapy. B4ECv3 as well as B2EC and B2EC-FC proteins blockedchemotaxis of human endothelial cells (as tested with umbilical cord andhepatic AECs or VECs), with a decrease in degradation of theextracellular matrix, Matrigel, and a decrease in migration in responseto growth factor stimuli (FIGS. 9-11). B4ECv3 and B2EC-FC proteins havepotent anti-angiogenic effect as demonstrated by their inhibition ofendothelial cell tube formation (FIGS. 12-13).

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

The sequence of the Globular domain+Cys-rich domain (B4EC-GC), precursorprotein is (SEQ ID NO:12):

MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAHHHHHH

For many uses, including therapeutic use, the leader sequence (first 15amino acids, so that the processed form begins Leu-Glu-Glu . . . ) andthe c-terminal hexahistidine tag may be removed or omitted.

Sequence of the GCF precursor protein (SEQ ID NO:13):

MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVHHHHHH

For many uses, including therapeutic use, the leader sequence (first 15amino acids, so that the processed form begins Leu-Glu-Glu . . . ) andthe c-terminal hexahistidine tag may be removed or omitted.

Amino acid sequence of encoded FL-hB4EC precursor (His-tagged) (SEQ IDNO:14):

MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLIFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQGSKRAILQIEGKP IPNPLLGLDSTRTGHHHHHH

For many uses, including therapeutic use, the leader sequence (first 15amino acids, so that the processed form begins Leu-Glu-Glu . . . ) andthe c-terminal hexahistidine tag may be removed or omitted.

EphB4 CF2 protein, precursor (SEQ ID NO:15):

MELRVLLCWASLAAALEETLLNTKLETQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQGGRSSLEGPRFEGKPIPNPLLGLDSTRTGHHHHH H

The precursor sequence of the preferred GCF2 protein (also referred toherein as GCF2F) is (SEQ ID NO:16):

MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQ

The processed sequence is (SEQ ID NO:17):

LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTYDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQ

Biochemical Assays

A. Binding Assay

10 μl of Ni-NTA-Agarose were incubated in microcentrifuge tubes with 50μl of indicated amount of B4ECv3 diluted in binding buffer BB (20 mMTris-HCl, 0.15 M NaCl, 0.1% bovine serum albumin pH 8) After incubationfor 30 min on shaking platform, Ni-NTA beads were washed twice with 1.4ml of BB, followed by application of 50 μl of B2-AP in the finalconcentration of 50 nM. Binding was performed for 30 min on shakingplatform, and then tubes were centrifuged and washed one time with 1.4ml of BB. Amount of precipitated AP was measured colorimetrically afterapplication of PNPP.

B. Inhibition Assay

Inhibition in solution. Different amounts of B4ECv3 diluted in 50 μl ofBB were pre-incubated with 50 μl of 5 nM B2EC-AP reagent (protein fusionof Ephrin B2 ectodomain with placental alkaline phosphatase). Afterincubation for 1 h, unbound B2EC-AP was precipitated with 5,000 HEK293cells expressing membrane-associated full-length EphB4 for 20 min.Binding reaction was stopped by dilution with 1.2 ml of BB, followed bycentrifugation for 10 min. Supernatants were discarded and alkalinephosphatase activities associated with collected cells were measured byadding para-nitrophenyl phosphate (PNPP) substrate.

Cell based inhibition. B4ECv3 was serially diluted in 20 mM Tris-HCl,0.15 M NaCl, 0.1% BSA, pH 8 and mixed with 5,000 HEK293 cells expressingmembrane-associated full-length Ephrin B2. After incubation for 1 h, 50μl of 5 nM B4EC-AP reagent (protein fusion of EphB4 ectodomain withplacental alkaline phosphatase were added into each tube for 30 min todetect unoccupied Ephrin B2 binding sites. Binding reactions werestopped by dilution with 1.2 ml of BB and centrifugation. Colorimetricreaction of cell-precipitated AP was developed with PNPP substrate.

C. B4EC-FC Binding Assay

Protein A-agarose based assay. 10 μl of Protein A-agarose were incubatedin Eppendorf tubes with 50 μl of indicated amount of B4EC-FC diluted inbinding buffer BB (20 mM Tris-HCl, 0.15 M NaCl, 0.1% BSA pH 8). Afterincubation for 30 min on shaking platform, Protein AAagarose beads werewashed twice with 1.4 ml of BB, followed by application of 50 μl ofB2ECAP reagent at the final concentration of 50 nM. Binding wasperformed for 30 min on shaking platform, and then tubes werecentrifuged and washed once with 1.4 ml of BB. Colorimetric reaction ofprecipitated AP was measured after application of PNPP (FIG. 6).

Nitrocellulose based assay. B4EC-FC was serially diluted in 20 mMTris-HCl, 0.15 M NaCl, 50 μg/ml BSA, pH 8. 2 μl of each fraction wereapplied onto nitrocellulose strip and spots were dried out for 3 min.Nitrocellulose strip was blocked with 5% non-fat milk for 30 min,followed by incubation with 5 nM B2EC-AP reagent. After 45 minincubation for binding, nitrocellulose was washed twice with 20 mMTris-HCl, 0.15 M NaCl, 50 μg/ml BSA, pH 8 and color was developed byapplication of alkaline phosphatase substrate Sigma Fast (Sigma).

D. B4EC-FC Inhibition Assay

Inhibition in solution. See above, for B4ECv3. The results were shown inFIG. 7.

Cell based inhibition. See above, for B4ECv3.

E. B2EC-FC Binding Assay

Protein-A-agarose based assay. See above, for B4EC-FC. The results wereshown in FIG. 8.

Nitrocellulose based assay. See above, for B4EC-FC.

6) Cell-Based Assays

A. Growth Inhibition Assay

Human umbilical cord vein endothelial cells (HUVEC) (1.5×103) are platedin a 96-well plate in 100 μl of EBM-2 (Clonetic # CC3162). After 24hours (day 0), the test recombinant protein (100 μl) is added to eachwell at 2× the desired concentration (5-7 concentration levels) in EBM-2medium. On day 0, one plate is stained with 0.5% crystal violet in 20%methanol for 10 minutes, rinsed with water, and air-dried. The remainingplates are incubated for 72 h at 37° C. After 72 h, plates are stainedwith 0.5% crystal violet in 20% methanol, rinsed with water andairdried. The stain is eluted with 1:1 solution of ethanol: 0.1 M sodiumcitrate (including day 0 plate), and absorbance is measured at 540 nmwith an ELISA reader (Dynatech Laboratories). Day 0 absorbance issubtracted from the 72 h plates and data is plotted as percentage ofcontrol proliferation (vehicle treated cells). IC50 (drug concentrationcausing 50% inhibition) is calculated from the plotted data.

B. Cord Formation Assay (Endothelial Cell Tube Formation Assay)

Matrigel (60 μl of 10 mg/ml; Collaborative Lab #35423) is placed in eachwell of an ice-cold 96-well plate. The plate is allowed to sit at roomtemperature for 15 minutes then incubated at 37° C. for 30 minutes topermit the matrigel to polymerize. In the mean time, HUVECs are preparedin EGM-2 (Clonetic #CC3162) at a concentration of 2×10⁵ cells/ml. Thetest compound is prepared at 2× the desired concentration (5concentration levels) in the same medium. Cells (500 μl) and 2× drug(500 μl) is mixed and 200 μl of this suspension are placed in duplicateon the polymerized matrigel. After 24 h incubation, triplicate picturesare taken for each concentration using a Bioquant Image Analysis system.Drug effect (IC50) is assessed compared to untreated controls bymeasuring the length of cords formed and number of junctions.

C. Cell Migration Assay

Migration is assessed using the 48-well Boyden chamber and 8 μm poresize collagen-coated (10 μg/ml rat tail collagen; CollaborativeLaboratories) polycarbonate filters (Osmonics, Inc.). The bottom chamberwells receive 27-29 μl of DMEM medium alone (baseline) or mediumcontaining chemo-attractant (bFGF, VEGF or Swiss 3T3 cell conditionedmedium). The top chambers receive 45 μl of HUVEC cell suspension (1×10⁶cells/ml) prepared in DMEM+1% BSA with or without test compound. After 5h incubation at 37° C., the membrane is rinsed in PBS, fixed and stainedin Diff-Quick solutions. The filter is placed on a glass slide with themigrated cells facing down and cells on top are removed using a Kimwipe.The testing is performed in 4-6 replicates and five fields are countedfrom each well. Negative unstimulated control values are subtracted fromstimulated control and drug treated values and data is plotted as meanmigrated cell±S.D. IC50 is calculated from the plotted data.

Example 2 Extracellular Domain Fragments of EphB4 Receptor InhibitAngiogenesis and Tumor Growth A. Globular Domain of EphB4 is Requiredfor EphrinB2 Binding and for the Activity of EphB4-Derived SolubleProteins in Endothelial Tube Formation Assay.

To identify subdomain(s) of the ectopic part of EphB4 necessary andsufficient for the anti-angiogenic activity of the soluble recombinantderivatives of the receptor, four recombinant deletion variants ofEphB4EC were produced and tested (FIG. 16). Extracellular part of EphB4,similarly to the other members of EphB and EphA receptor family,contains N-terminal ligand-binding globular domain followed bycysteine-rich domain and two fibronectin type III repeats (FNIII). Inaddition to the recombinant B4-GCF2 protein containing the completeectopic part of EphB4, we constructed three deletion variants of EphB4ECcontaining globular domain and Cys-rich domain (B4-GC); globular,Cys-rich and the first FNIII domain (GCF1) as well as the ECD versionwith deleted globular domain (CF2). Our attempts to produce severalversions of truncated EphB4EC protein containing the globular domainalone were not successful due to the lack of secretion of proteinsexpressed from all these constructs and absence of ligand binding by theintracellularly expressed recombinant proteins. In addition, anon-tagged version of B4-GCF2, called GCF2-F, containing completeextracellular domain of EphB4 with no additional fused amino acids wasexpressed, purified and used in some of the experiments described here.

All four C-terminally 6×His tagged recombinant proteins werepreparatively expressed in transiently transfected cultured mammaliancells and affinity purified to homogeneity from the conditioned growthmedia using chromatography on Ni²⁺-chelate resin (FIG. 17). Apparentlydue to their glycosylation, the proteins migrate on SDS-PAAG somewhathigher than suggested by their predicted molecular weights of 34.7 kDa(GC), 41.5 (CF2), 45.6 kDa (GCF1) and 57.8 kDa (GCF2). Sequence of theextracellular domain of human EphB4 contains three predictedN-glycosylation sites (NXS/T) which are located in the Cys-rich domain,within the first fibronectin type III repeat and between the first andthe second fibronectin repeats.

To confirm ability of the purified recombinant proteins to bind EphrinB2, they were tested in an in vitro binding assay. As expected, GC, GCF1and GCF2, but not CF2 are binding the cognate ligand Ephrin B2 asconfirmed by interaction between Ephrin B2-alkaline phosphatase (EphrinB2-AP) fusion protein with the B4 proteins immobilized on Ni²⁺-resin oron nitrocellulose membrane (FIG. 17).

All four proteins were also tested for their ability to blockligand-dependent dimerization and activation of Eph B4 receptor kinasein PC3 cells. The PC3 human prostate cancer cell line is known toexpress elevated levels of human Eph B4. Stimulation of PC3 cells withEphrin B2 IgG Fc fusion protein leads to a rapid induction of tyrosinephosphorylation of the receptor. However, preincubation of the ligandwith GCF2, GCF1 or GC, but not CF2 proteins suppresses subsequent EphB4autophosphorylation. Addition of the proteins alone to the PC3 cells orpreincubation of the cells with the proteins followed by changing mediaand adding the ligand does not affect EphB4 phosphorylation status.

Further, we found that globular domain of EphB4 is required for theactivity of EphB4-derived soluble proteins in endothelial tube formationassay.

B. Effects of Soluble EphB4 on HUV/AEC In Vitro.

Initial experiments were performed to determine whether soluble EphB4affected the three main stages in the angiogenesis pathway. These werecarried out by establishing the effects of soluble EphB4 onmigration/invasion, proliferation and tubule formation by HUV/AEC invitro. Exposure to soluble EphB4 significantly inhibited both bFGF andVEGF-induced migration in the Boyden chamber assay in a dose-dependentmanner, achieving significance at nM (FIG. 18). Tubule formation byHUV/AECS on wells coated with Matrigel was significantly inhibited bysoluble EphB4 in a dose-dependent manner in both the absence andpresence of bFGF and VEGF (FIG. 19). We also assessed in vitro, whethernM of soluble EphB4 was cytotoxic for HUVECS. Soluble EphB4 was found tohave no detectable cytotoxic effect at these doses, as assessed by MTSassay (FIG. 20).

C. Soluble EphB4 Receptor Inhibits Vascularization of Matrigel Plugs, InVivo

To demonstrate that soluble EphB4 can directly inhibit angiogenesis invivo, we performed a murine matrigel plug experiment. Matrigelsupplemented with bFGF and VEGF with and without soluble EphB4 wasinjected s.c. into Balb/C nu/nu mice, forming semi-solid plugs, for sixdays. Plugs without growth factors had virtually no vascularization orvessel structures after 6 days (FIG. 21). In contrast, plugssupplemented with bFGF and VEGF had extensive vascularization andvessels throughout the plug. Plugs taken from mice treated with μg ofsoluble EphB4 had markedly reduced vascularization of plugs, comparableto plugs without growth factor (FIG. 21). Furthermore, histologicalexamination of plugs showed decreased vessel staining (FIG. 21).Treatment at 0 μg/dose significantly inhibited the amount ofinfiltration in Matrigel plugs compared to control (FIG. 21).

We examined EphB4 receptor phosphorylation in HUVECs by performingWestern blot analyses with lysates from soluble EphB4-treated cells andantibodies against phosphor-tyrosine. We found that soluble EphB4treatment of serum-starved HUVECs stimulated a rapid and transientdecrease in the level of phosphorylated EphB4, in the presence ofEphrinB2Fc, EphB4 ligand dimer. Ephrin B2Fc without the soluble EphB4protein induced phosphorylation of EphB4 receptor (FIG. 22).

D. Effects of Soluble EphB4 on Tumor Growth, In Vitro.

We found that soluble EphB4 inhibits the growth of SCC15 tumors grown inBalb/C Nu/Nu mice (FIG. 23).

E. Soluble EphB4 Inhibited Corneal Neovascularization

To further investigate the antiangiogenic activity of soluble EphB4 invivo, we studied the inhibitory effect of administration of solubleEphB4 on neovascularization in the mouse cornea induced by bFGF. HydronPellets implanted into corneal micropocket could induce angiogenesis, inthe presence of growth factors, in a typically avascular area. Theangiogenesis response in mice cornea was moderate, the appearance ofvascular buds was delayed and the new capillaries were sparse and grewslowly. Compared with the control group, on day 7 of implantation, theneovascularization induced by bFGF in mice cornea was markedly inhibitedin soluble EphB4-treated group (FIG. 24).

F. Effects of Soluble EphB4 on Tumor Growth, In Vivo.

The same model was used to determine the effects of soluble EphB4 invivo. SCC15 tumors implanted subcutaneously, pre-incubated with matrigeland with or w/o growth factors, as well as implanted sc alone, and micetreated sc or ip daily with 1-5 ug of soluble EphB4 were carried out.

Tumors in the control group continued to grow steadily over thetreatment period, reaching a final tumor volume of mm3. However, animalsinjected with soluble EphB4 exhibited a significantly (p<0.0/) reducedgrowth rate, reaching a final tumor volume of only mm3 (FIG. 25).Similar results were obtained in two further cohorts of suchtumor-bearing mice. Soluble EphB4 administration appeared to be welltolerated in vivo, with no significant effect on body weight or thegeneral well-being of the animals (as determined by the absence oflethargy, intermittent hunching, tremors or disturbed breathingpatterns).

G. Effects of Soluble EphB4 on Tumor Histology.

Histological analysis revealed the presence of a central area ofnecrosis in all SCC15 tumors, which was usually surrounded by a viablerim of tumor cells um in width. The central necrotic areas werefrequently large and confluent and showed loss of cellular detail.Necrosis, assessed as a percentage of tumor section area, wassignificantly (p<0.02) more extensive in the soluble EphB4-treated group(% necrosis in treated vs. control). To determine whether the reducedvolume of soluble EphB4 treated tumors was due to an effect of thisprotein on the tumor vascular supply, endothelial cells in blood vesselswere identified in tumor sections using immunostaining with ananti-platelet cell adhesion molecule (PECAM-1; CD31) antibody (FIG. 26)and the density of microvessels was assessed. Microvessel density wassimilar in the outer viable rim of tumor cells (the uniform layer ofcells adjacent to the tumor periphery with well defined nuclei) incontrol and soluble EphB4-treated tumors. Microvessel density wassignificantly in the inner, less viable region of tumor cells abuttingthe necrotic central areas in soluble EphB4-treated than control tumors.Fibrin deposition, as identified by Masson's Trichrome staining, wasincreased in and around blood vessels in the inner viable rim and thecentral necrotic core of soluble EphB4 treated than control tumors. Inthe outer viable rim of soluble EphB4 treated tumors, although thevessel lumen remained patent and contained red blood cells, fibrindeposition was evident around many vessels. Soluble EphB4 was found tohave no such effects on the endothelium in the normal tissues examined(lungs, liver and kidneys).

H. Materials and Methods

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

Cell-Based EphB4 Tyrosine Kinase Assay

The human prostate carcinoma cell line PC3 cells were maintained in RPMImedium with 10% dialyzed fetal calf serum and 1%penicillin/streptomycin/neomycin antibiotics mix. Cells were maintainedat 37° C. in a humidified atmosphere of 5% CO₂/95% air. Typically, cellswere grown in 60 mm dishes until confluency and were either treated withmouse Ephrin B2-Fc fusion at 1 μg/ml in RPMI for 10 min to activateEphB4 receptor or plain medium as a control. To study the effect ofdifferent derivatives of soluble EphB4 ECD proteins on EphB4 receptoractivation, three sets of cells were used. In the first set, cells weretreated with various proteins (5 proteins; GC, GCF1, GCF2, GCF2-F, CF2)at 5 μg/ml for 20 min. In the second set of cells, prior to application,proteins were premixed with ephrinB2-Fc at 1:5 (EphB4 protein:B2-Fc)molar ratio, incubated for 20 min and applied on cells for 10 min. Inthe third set of cells, cells were first treated with the proteins for20 min at 5 μg/ml, media was replaced with fresh media containing 1μg/ml of EphrinB2-Fc and incubated for another 10 min.

After the stimulation, cells were immediately harvested with proteinextraction buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%(v/v) Triton X100, 1 mM EDTA, 1 mM PMSF, 1 mM Sodium vanadate. Proteinextracts were clarified by centrifugation at 14,000 rpm for 20 min at 4°C. Clarified protein samples were incubated overnight with protein A/Gcoupled agarose beads pre-coated with anti-EphB4 monoclonal antibodies.The IP complexes were washed twice with the same extraction buffercontaining 0.1% Triton X100. The immunoprecipitated proteins weresolubilized in 1×SDS-PAGE sample loading buffer and separated on 10%SDS-PAGE. For EphB4 receptor activation studies, electroblotted membranewas probed with anti-pTyr specific antibody 4G10 at 1:1000 dilutionfollowed by Protein G-HRP conjugate at 1:5000 dilutions.

Endothelial Cell Tube Formation Assay

Matrigel (60 μl of 10 mg/ml; Collaborative Lab, Cat. No. 35423) wasplaced in each well of an ice-cold 96-well plate. The plate was allowedto sit at room temperature for 15 minutes then incubated at 37° C. for30 minutes to permit Matrigel to polymerize. In the mean time, humanumbilical vein endothelial cells were prepared in EGM-2 (Clonetic, Cat.No. CC3162) at a concentration of 2×10⁵ cells/ml. The test protein wasprepared at 2× the desired concentration (5 concentration levels) in thesame medium. Cells (500 μl) and 2× protein (500 μl) were mixed and 200μl of this suspension were placed in duplicate on the polymerizedMatrigel. After 24 h incubation, triplicate pictures were taken for eachconcentration using a Bioquant Image Analysis system. Protein additioneffect (IC₅₀) was assessed compared to untreated controls by measuringthe length of cords formed and number of junctions.

Cell Migration Assay

Chemotaxis of HUVECs to VEGF was assessed using a modified Boydenchamber, transwell membrane filter inserts in 24 well plates, 6.5 mmdiam, 8 μm pore size, 10 μm thick matrigel coated, polycarbonatemembranes (BD Biosciences). The cell suspensions of HUVECs (2×10⁵cells/ml) in 200 μl of EBM were seeded in the upper chamber and thesoluble EphB4 protein were added simultaneously with stimulant (VEGF orbFGF) to the lower compartment of the chamber and their migration acrossa polycarbonate filter in response to 10-20 ng/ml of VEGF with orwithout 100 nM-1 μM test compound was investigated. After incubation for4-24 h at 37° C., the upper surface of the filter was scraped with swaband filters were fixed and stained with Diff Quick. Ten random fields at200× mag were counted and the results expressed as mean # per field.Negative unstimulated control values were subtracted from stimulatedcontrol and protein treated sample values and the data was plotted asmean migrated cell±S.D. IC₅₀ was calculated from the plotted data.

Growth Inhibition Assay

HUVEC (1.5×10³ cells) were plated in a 96-well plate in 100 μl of EBM-2(Clonetic, Cat. No. CC3162). After 24 hours (day 0), the testrecombinant protein (100 μl) is added to each well at 2× the desiredconcentration (5-7 concentration levels) in EBM-2 medium. On day 0, oneplate was stained with 0.5% crystal violet in 20% methanol for 10minutes, rinsed with water, and air-dried. The remaining plates wereincubated for 72 h at 37° C. After 72 h, plates were stained with 0.5%crystal violet in 20% methanol, rinsed with water and air-dried. Thestain was eluted with 1:1 solution of ethanol: 0.1M sodium citrate(including day 0 plate), and absorbance measured at 540 nm with an ELISAreader (Dynatech Laboratories). Day 0 absorbance was subtracted from the72 h plates and data is plotted as percentage of control proliferation(vehicle treated cells). IC₅₀ value was calculated from the plotteddata.

Murine Matrigel Plug Angiogenesis Assay

In vivo angiogenesis was assayed in mice as growth of blood vessels fromsubcutaneous tissue into a Matrigel plug containing the test sample.Matrigel rapidly forms a solid gel at body temperature, trapping thefactors to allow slow release and prolonged exposure to surroundingtissues. Matrigel (8.13 mg/ml, 0.5 ml) in liquid form at 4° C. was mixedwith Endothelial Cell Growth Supplement (ECGS), test proteins plus ECGSor Matrigel plus vehicle alone (PBS containing 0.25% BSA). Matrigel (0.5ml) was injected into the abdominal subcutaneous tissue of female nu/numice (6 wks old) along the peritoneal mid line. There were 3 mice ineach group. The animals were cared for in accordance with institutionaland NIH guidelines. At day 6, mice were sacrificed and plugs wererecovered and processed for histology. Typically the overlying skin wasremoved, and gels were cut out by retaining the peritoneal lining forsupport, fixed in 10% buffered formalin in PBS and embedded in paraffin.Sections of 3 μm were cut and stained with H&E or Masson's trichromestain and examined under light microscope

Mouse Corneal Micropocket Assay

Mouse corneal micropocket assay was performed according to that detailedby Kenyon et al., 1996. Briefly, hydron pellets(polyhydroxyethylmethacrylate [polyHEMA], Interferon Sciences, NewBrunswick, N.J., U.S.A.) containing either 90 ng of bFGF (R&D) or 180 ngof VEGF (R&D Systems, Minneapolis, Minn., U.S.A.) and 40 μg of sucrosealuminium sulfate (Sigma) were prepared. Using an operating microscope,a stromal linear keratotomy was made with a surgical blade (Bard-Parkerno. 15) parallel to the insertion of the lateral rectus muscle in ananesthetized animal. An intrastromal micropocket was dissected using amodified von Graefe knife (2″30 mm). A single pellet was implanted andadvanced toward the temporal corneal limbus (within 0±7±1±0 mm for bFGFpellets and 0±5 mm for VEGF pellets). The difference in pellet locationfor each growth factor was determined to be necessary given therelatively weaker angiogenic stimulation of VEGF in this model.Antibiotic ointment (erythromycin.) was then applied to the operated eyeto prevent infection and to decrease surface irregularities. Thesubsequent vascular response was measured extending from the limbalvasculature toward the pellet and the contiguous circumferential zone ofneovascularization Data and clinical photos presented here were obtainedon day 6 after pellet implantation, which was found to be the day ofmaximal angiogenic response.

In Vitro Invasion Assay

“Matrigel” matrix-coated 9-mm cell culture inserts (pore size, 8 μm;Becton Dickinson, Franklin Lakes, N.J.) were set in a 24-well plate. TheHUVEC cells were seeded at a density of 5×10³ cells per well into theupper layer of the culture insert and cultured with serum-free EBM inthe presence of EphB4 ECD for 24 h. The control group was cultured inthe same media without EphB4. Then 0.5 ml of the human SCC15 cell line,conditioned medium was filled into the lower layer of the culture insertas a chemo-attractant. The cells were incubated for 24 h, then theremaining cells in the upper layer were swabbed with cotton andpenetrating cells in the lower layer were fixed with 5% glutaraldehydeand stained with Diff Quick. The total number of cells passing throughthe Matrigel matrix and each 8 μm pore of the culture insert was countedusing optical microscopy and designated as an invasion index (cellnumber/area).

SCC15 Tumor Growth in Mice

Subcutaneously inject logarithmically growing SCC15, head and necksquamous cell carcinoma cell line, at 5×10⁶ cell density; with orwithout EphB4 ECD in the presence or absence of human bFGF, into athymicBalb/c nude mice, along with Matrigel (BD Bioscience) synthetic basementmembrane (1:1 v/v), and examine tumors within 2 weeks. Tumor volumes inthe EphB4 ECD group, in the presence and absence of growth factor afterimplantation were three-fold smaller than those in the vehicle groups.There was no difference in body weight between the groups.Immunohistochemical examination of cross-sections of resected tumors andTUNEL-positive apoptosis or necrosis, CD34 immunostaining, and BrdUproliferation rate will be performed, after deparaffinized, rehydrated,and quenched for endogenous peroxidase activity, and after 10 minpermeabilization with proteinase K. Quantitative assessment of vasculardensities will also be performed. Local intratumoral delivery or IVdelivery of EphB4 ECD will also be performed twice a week.

30 athymic nude mice, BALB/c (nu/nu), were each injected with 1×10⁶ B16melanoma cells with 0.1 ml PBS mixed with 0.1 ml matrigel or 1.5×10⁶SCC15 cells resuspended in 200 μl of DMEM serum-free medium and injectedsubcutaneously on day 0 on the right shoulder region of mice. Proteinswere injected intravenously or subcutaneously, around the tumorbeginning on day 1 at a loading dose of 4 μg/mg, with weekly injectionsof 2 ug/mg. (10 μg/g, 50 μg/kg/day), and at 2 weeks post-inoculation.Mice are sacrificed on Day 14. Control mice received PBS 50 μl each day.

Tumor Formation in Nude Mice

All animals were treated under protocols approved by the institutionalanimal care committees. Cancer cells (5×10⁶) were subcutaneouslyinoculated into the dorsal skin of nude mice. When the tumor had grownto a size of about 100 mm³ (usually it took 12 days), sEphB4 was eitherintraperitoneally or subcutaneously injected once/day, and tumorigenesiswas monitored for 2 weeks. Tumor volume was calculated according to theformula a²×b, where a and b are the smallest and largest diameters,respectively. A Student's t test was used to compare tumor volumes, withP<0.05 being considered significant.

Quantification of Microvessel Density

Tumors were fixed in 4% formaldehyde, embedded in paraffin, sectioned by5 μm, and stained with hematoxylineosin. Vessel density wassemi-quantitated using a computer-based image analyzer (five fields persection from three mice in each group).

Example 3 EphB4 is Upregulated and Imparts Growth Advantage in ProstateCancer A. Expression of EphB4 in Prostate Cancer Cell Lines

We first examined the expression of EphB4 protein in a variety ofprostate cancer cell lines by Western blot. We found that prostatecancer cell lines show marked variation in the abundance of the 120 kDEphB4. The levels were relatively high in PC3 and even higher in PC3M, ametastatic clone of PC3, while normal prostate gland derived cell lines(MLC) showed low or no expression of EphB4 (FIG. 27A). We next checkedthe activation status of EphB4 in PC3 cells by phosphorylation study. Wefound that even under normal culture conditions, EphB4 is phosphorylatedthough it can be further induced by its ligand, ephrin B2 (FIG. 27B).

B. Expression of EphB4 in Clinical Prostate Cancer Samples

To determine whether EphB4 is expressed in clinical prostate samples,tumor tissues and adjacent normal tissue from prostate cancer surgicalspecimens were examined. The histological distribution of EphB4 in theprostate specimens was determined by immunohistochemistry. Clearly,EphB4 expression is confined to the neoplastic epithelium (FIG. 28, topleft), and is absent in stromal and normal prostate epithelium (FIG. 28,top right). In prostate tissue array, 24 of the 32 prostate cancersexamined were positive. We found EphB4 mRNA is expressed both in thenormal and tumor tissues of clinical samples by quantitative RT-PCR.However, tumor EphB4 mRNA levels were at least 3 times higher than inthe normal in this case (FIG. 28, lower right).

C. p53 and PTEN Inhibited the Expression of EphB4 in PC3 Cells

PC3 cells are known to lack PTEN expression (Davis, et al., 1994,Science. 266:816-819) and wild-type p53 function (Gale, et al., 1997,Cell Tissue Res. 290:227-241). We investigated whether the relativelyhigh expression of EphB4 is related to p53 and/or PTEN by re-introducingwild-type p53 and/or PTEN into PC3 cells. To compensate for thetransfection efficiency and the dilution effect, transfected cells weresorted for the cotransfected truncated CD4 marker. We found that theexpression of EphB4 in PC3 cells was reduced by the re-introduction ofeither wild-type p53 or PTEN. The co-transfection of p53 and PTEN didnot further inhibit the expression of EphB4 (FIG. 29A).

D. Retinoid X Receptor (RXR α) Regulates the Expression of EphB4

We previously found that RXRα was down-regulated in prostate cancer celllines (Thong, et al., 2003, Cancer Biol Ther. 2:179-184) and here wefound EphB4 expression has the reverse expression pattern when we lookedat “normal” prostate (MLC), prostate cancer (PC3), and metastaticprostate cancer (PC3M) (FIG. 27A), we considered whether RXRα regulatesthe expression of EphB4. To confirm the relationship, the expression ofEphB4 was compared between CWR22R and CWR22R-RXRα, which constitutivelyexpresses RXRα. We found a modest decrease in EphB4 expression in theRXRα overexpressing cell line, while FGF8 has no effect on EphB4expression. Consistent with initial results, EphB4 was not found in“normal” benign prostate hypertrophic cell line BPH-1 (FIG. 29B).

E. Growth Factor Signaling Pathway of EGFR and IGF-1R Regulates EphB4Expression

EGFR and IGF-1R have both been shown to have autocrine and paracrineaction on PC3 cell growth. Because we found that EphB4 expression ishigher in the more aggressive cell lines, we postulated that EphB4expression might correlate with these pro-survival growth factors. Wetested the relationship by independently blocking EGFR and IGF-1Rsignaling. EphB4 was down-regulated after blocking the EGFR signalingusing EGFR kinase inhibitor AG 1478 (FIG. 30A) or upon blockade of theIGF-1R signaling pathway using IGF-1R neutralizing antibody (FIG. 30B).

F. EphB4 siRNA and Antisense ODNs Inhibit PC3 Cell Viability

To define the significance of this EphB4 overexpression in our prostatecancer model, we concentrated our study on PC3 cells, which have arelatively high expression of EphB4. The two approaches to decreasingEphB4 expression were siRNA and AS-ODNs. A number of differentphosphorothioate-modified AS-ODNs complementary to different segments ofthe EphB4 coding region were tested for specificity and efficacy ofEphB4 inhibition. Using 293 cells transiently transfected withfull-length EphB4 expression vector AS-10 was found to be the mosteffective (FIG. 31B). A Similar approach was applied to the selection ofspecific siRNA. EphB4 siRNA 472 effectively knocks down EphB4 proteinexpression (FIG. 31A). Both siRNA 472 and antisense AS-10 ODN reducedthe viability of PC3 cells in a dose dependent manner (FIGS. 31C, D).Unrelated siRNA or sense oligonucleotide had no effect on viability.

G. EphB4 siRNA and Antisense ODNs Inhibit the Mobility of PC3 Cells

PC3 cells can grow aggressively locally and can form lymph nodemetastases when injected orthotopically into mice. In an effort to studythe role of EphB4 on migration of PC3 cells in vitro, we performed awound-healing assay. When a wound was introduced into a monolayer of PC3cells, over the course of the next 20 hours cells progressively migratedinto the cleared area. However, when cells were transfected with siRNA472 and the wound was introduced, this migration was significantlyinhibited (FIG. 31E). Pretreatment of PC3 cells with 10 μM EphB4 AS-10for 12 hours generated the same effect (FIG. 31F). In addition,knock-down of EphB4 expression in PC3 cells with siRNA 472 severelyreduced the ability of these cells to invade Matrigel as assessed by adouble-chamber invasion assay (FIG. 31G), compared to the control siRNA.

H. EphB4 siRNA Induces Cell Cycle Arrest and Apoptosis in PC3 Cells

Since knock-down of EphB4 resulted in decreased cell viability (FIG.31C) we sought to determine whether this was due to effects on the cellcycle. In comparison to control siRNA transfected cells, siRNA 472resulted in an accumulation of cells in the sub G0 and S phase fractionscompared to cells treated with control siRNA. The sub G0 fractionincreased from 1% to 7.9%, and the S phase fraction from 14.9% to 20.8%in siRNA 472 treated cells compared to control siRNA treated cells (FIG.32A). Cell cycle arrest at sub G0 and G2 is indicative of apoptosis.Apoptosis as a result of EphB4 knock-down was confirmed by ELISA assay.A dose-dependent increase in apoptosis was observed when PC3 cells weretransfected with siRNA 472, but not with control siRNA (FIG. 32B). At100 nM there was 15 times more apoptosis in siRNA 472 transfected thancontrol siRNA transfected PC3 cells.

I. Materials and Methods

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

Example 4 Expression of EPHB4 in Mesothelioma: a Candidate Target forTherapy

Malignant mesothelioma (MM) is a rare neoplasm that most often arisesfrom the pleural and peritoneal cavity serous surface. The pleuralcavity is by far the most frequent site affected (>90%), followed by theperitoneum (6-10%) (Carbone et al., 2002, Semin Oncol. 29:2-17). Thereis a strong association with asbestos exposure, about 80% of malignantmesothelioma cases occur in individuals who have ingested or inhaledasbestos. This tumor is particularly resistant to the current therapiesand, up to now, the prognosis of these patients is dramatically poor(Lee et al., 2000, Curr Opin Pulm Med. 6:267-74).

Several clinical problems regarding the diagnosis and treatment ofmalignant mesothelioma remain unsolved. Making a diagnosis ofmesothelioma from pleural or abdominal fluid is notoriously difficultand often requires a thoracoscopic or laproscopic or open biopsy andImmunohistochemical staining for certain markers such as meosthelinexpressed preferentially in this tumor. Until now, no intervention hasproven to be curative, despite aggressive chemotherapeutic regimens andprolonged radiotherapy. The median survival in most cases is only 12-18months after diagnosis.

In order to identify new diagnostic markers and targets to be used fornovel diagnostic and therapeutic approaches, we assessed the expressionof EPHB4 and its ligand EphrinB2 in mesothelioma cell lines and clinicalsamples.

A. EPHB4 and EphrinB2 is Expressed in Mesothelioma Cell Lines

The expression of Ephrin B2 and EphB4 in malignant mesothelioma celllines was determined at the RNA and protein level by a variety ofmethods. RT-PCR showed that all of the four cell lines express EphrinB2and EPHB4 (FIG. 33A). Protein expression was determined by Western blotin these cell lines. Specific bands for EphB4 were seen at 120 kD. Inaddition, Ephrin B2 was detected in all cell lines tested as a 37 kDband on Western blot (FIG. 33B). No specific band for Ephrin B2 wasobserved in 293 human embryonic kidney cells, which were included as anegative control.

To confirm the presence of EphB4 transcription in mesothelioma cells, insitu hybridization was carried out on NCI H28 cell lines cultured onchamber slides. Specific signal for EphB4 was detected using antisenseprobe Ephrin B2 transcripts were also detected in the same cell line.Sense probes for both EphB4 and Ephrin B2 served as negative controlsand did not hybridize to the cells (FIG. 34). Expression of EphB4 andEphrin B2 proteins was confirmed in the cell lines by immunofluorescenceanalysis (FIG. 35). Three cell lines showed strong expression of EphB4,whereas expression of Ephrin B2 was present in H28 and H2052, and weaklydetectable in H2373.

B. Evidence of Expression of EPHB4 and EphrinB2 in Clinical Samples

Tumor cells cultured from the pleural effusion of a patient diagnosedwith pleural malignant mesothelioma were isolated and showed positivestaining for both EphB4 and Ephrin B2 at passage 1 (FIG. 35, bottomrow). These results confirm co-expression of EphB4 and Ephrin B2 inmesothelioma cell lines. To determine whether these results seen intumor cell lines were a real reflection of expression in the diseasestate, tumor biopsy samples were subjected to immunohistochemicalstaining for EphB4 and Ephrin B2. Antibodies to both proteins revealedpositive stain in the tumor cells. Representative data is shown in FIG.36.

C. EPHB4 is Involved in the Cell Growth and Migration of Mesothelioma

The role of EphB4 in cell proliferation was tested using EPHB4 specificantisepses oligonucleotides and siRNA. The treatment of cultured H28with EPHB4 antisense reduced cell viability. One of the most activeinhibitor of EphB4 expression is EPHB4AS-10 (FIG. 37A). Transfection ofEPHB4 siRNA 472 generated the same effect (FIG. 37B).

MM is a locally advancing disease with frequent extension and growthinto adjacent vital structures such as the chest wall, heart, andesophagus. In an effort to study this process in vitro, we perform woundhealing assay using previously described techniques (3:36). When a woundwas introduced into sub confluent H28 cells, over the course of the next28 hours cells would progressively migrate into the area of the wound.However, when cells were pretreated with EPHB4AS-10 for 24 hours, andthe wound was introduced, this migration was virtually completelyprevented (FIG. 38A). The migration study with Boyden Chamber assay withEPHB4 siRNA showed that cell migration was greatly inhibited with theinhibition of EPHB4 expression (FIG. 38B).

D. Materials and Methods

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

Example 5 EphB4 is Expressed in Squamous Cell Carcinoma of The Head andNeck: Regulation by Epidermal Growth Factor Signaling Pathway and GrowthAdvantage

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth mostfrequent cancer worldwide, with estimated 900,000 cases diagnosed eachyear. It comprises almost 50% of all malignancies in some developingnations. In the United States, 50,000 new cases and 8,000 deaths arereported each year. Tobacco carcinogens are believed to be the primaryetiologic agents of the disease, with alcohol consumption, age, gender,and ethnic background as contributing factors.

The differences between normal epithelium of the upper aerodigestivetract and cancer cells arising from that tissue are the result ofmutations in specific genes and alteration of their expression. Thesegenes control DNA repair, proliferation, immortalization, apoptosis,invasion, and angiogenesis. For head and neck cancer, alterations ofthree signaling pathways occur with sufficient frequency and producesuch dramatic phenotypic changes as to be considered the criticaltransforming events of the disease. These changes include mutation ofthe p53 tumor suppressor, overexpression of epidermal growth factorreceptor (EGFR), and inactivation of the cyclin dependent kinaseinhibitor p16. Other changes such as Rb mutation, ras activation, cyclinD amplification, and myc overexpression are less frequent in HNSCC.

Although high expression of EphB4 has been reported in hematologicmalignancies, breast carcinoma, endometrial carcinoma, and coloncarcinoma, there is limited data on the protein levels of EphB4, andcomplete lack of data on the biological significance of this protein intumor biology such as HNSCC.

A. HNSCC Tumors Express EphB4

We studied the expression of EphB4 in human tumor tissues byimmunohistochemistry, in situ hybridization, and Western blot. Twentyprospectively collected tumor tissues following IRB approval have beenevaluated with specific EphB4 monoclonal antibody that does not reactwith other members of the EphB and EphA family. EphB4 expression isobserved in all cases, with varying intensity of staining. FIG. 39A (topleft) illustrates a representative case, showing that EphB4 is expressedin the tumor regions only, as revealed by the H&E tumor architecture(FIG. 39A bottom left). Note the absence of staining for EphB4 in thestroma. Secondly, a metastatic tumor site in the lymph node showspositive staining while the remainder of the lymph node is negative(FIG. 39A, top right).

In situ hybridization was carried out to determine the presence andlocation of EphB4 transcripts in the tumor tissue. Strong signal forEphB4 specific antisense probe was detected indicating the presence oftranscripts (FIG. 39 B, top left). Comparison with the H&E stain (FIG.39B, bottom left) to illustrate tumor architecture reveals that thesignal was localized to the tumor cells, and was absent from the stromalareas. Ephrin B2 transcripts were also detected in tumor sample, and aswith EphB4, the signal was localized to the tumor cells (FIG. 39B, topright). Neither EphB4 nor ephrin B2 sense probes hybridized to thesections, proving specificity of the signals.

B. High Expression of EphB4 in Primary and Metastatic Sites of HNSCC

Western blots of tissue from primary tumor, lymph node metastases anduninvolved tissue were carried out to determine the relative levels ofEphB4 expression in these sites. Tumor and normal adjacent tissues werecollected on 20 cases, while lymph nodes positive for tumor wereharvested in 9 of these 20 cases. Representative cases are shown in FIG.39C. EphB4 expression is observed in each of the tumor samples.Similarly, all tumor positive lymph nodes show EphB4 expression that wasequal to or greater than the primary tumor. No or minimal expression isobserved in the normal adjacent tissue.

C. EphB4 Expression and Regulation by EGFR Activity in HNSCC Cell Lines

Having demonstrated the expression of EphB4 limited to tumor cells, wenext sought to determine whether there was an in vitro model of EphB4expression in HNSCC. Six HN SCC cell lines were surveyed for EphB4protein expression by Western Blot (FIG. 40A). A majority of theseshowed strong EphB4 expression and thus established the basis forsubsequent studies. Since EGFR is strongly implicated in HNSCC we askedwhether EphB4 expression is associated with the activation of EGFR.Pilot experiments in SCC-15, which is an EGFR positive cell line,established an optimal time of 24 h and concentration of 1 mM of thespecific EGFR kinase inhibitor AG 1478 (FIG. 40B) to inhibit expressionof EphB4. When all the cell lines were studied, we noted robust EGFRexpression in all but SCC-4, where it is detectable but not strong (FIG.40C, top row). In response to EGFR inhibitor AG1478 marked loss in thetotal amount of EphB4 was observed in certain cell lines (SCC-15, andSCC-25) while no effect was observed in others (SCC-9, -12, -13 and-71). Thus SCC-15 and -25 serve as models for EphB4 being regulated byEGFR activity, while SCC-9, -12, -13 and -71 are models for regulationof EphB4 in HNSCC independent of EGFR activity, where there may be inputfrom other factors such as p53, PTEN, IL-6 etc. We also noted expressionof the ligand of EphB4, namely ephrin B2, in all of the cell linestested. As with EphB4 in some lines ephrin B2 expression appearsregulated by EGFR activity, while it is independent in other cell lines.

Clearly, inhibition of constitutive EGFR signaling repressed EphB4levels in SCC15 cells. We next studied whether EGF could induce EphB4.We found that EphB4 levels were induced in SCC15 cells that had beenserum starved for 24 h prior to 24 h treatment with 10 ng/ml EGF asshown in FIG. 41B (lanes 1 and 2). The downstream signaling pathwaysknown for EGFR activation shown in FIG. 41A, (for review see Yarden &Slikowski 2001) were then investigated for their input into EGF mediatedinduction of EphB4. Blocking PLCg, AKT and JNK phosphorylation with thespecific kinase inhibitors U73122, SH-5 and SP600125 respectivelyreduced basal levels and blocked EGF stimulated induction of EphB4 (FIG.41B, lanes 3-8). In contrast, inhibition of ERK1/2 with PD098095 andPI3-K with LY294002 or Wortmannin had no discernible effect on EGFinduction of EphB4 levels. However, basal levels of EphB4 were reducedwhen ERK1/2 phosphorylation was inhibited. Interestingly, inhibition ofp38 MAPK activation with SB203580 increased basal, but not EGF inducedEphB4 levels. Similar results were seen in the SCC25 cell line (data notshown).

D. Inhibition of EphB4 in High Expressing Cell Lines Results in ReducedViability and Causes Cell-Cycle Arrest

We next turned to the role of EphB4 expression in HNSCC by investigatingthe effect of ablating expression using siRNA or AS-ODN methods. SeveralsiRNAs to EphB4 sequence were developed (Table 1) which knocked-downEphB4 expression to varying degrees as seen in FIG. 42A. Viability wasreduced in SCC-15, -25 and -71 cell lines transfected with siRNAs 50 and472, which were most effective in blocking EphB4 expression (FIG. 42B).Little effect on viability was seen with EphB4 siRNA 1562 and 2302 orephrin B2 siRNA 254. Note that in SCC-4, which does not express EphB4(see FIG. 40A) there was no reduction in cell viability. The decreasedcell viability seen with siRNA 50 and 472 treatment was attributable toaccumulation of cells in sub G0, indicative of apoptosis. This effectwas both time and dose-dependant (FIG. 42C and Table 2). In contrast,siRNA2302 that was not effective in reducing EphB4 levels and had onlyminor effects on viability did not produce any changes in the cell cyclewhen compared with the mock Lipofectamine™2000 transfection.

A detailed description of the siRNA constructs for this example may befound in U.S. Patent Publication No. 20050084873.

TABLE Effect of different EphB4 siRNA on Cell Cycle Treatment Sub G0 G1S G2 36 hr Lipo alone 1.9 39.7 21.3 31.8 100 nM 2302 2.0 39.3 21.2 31.2100 nM 50 18.1 31.7 19.7 24.4 100 nM 472 80.2 10.9 5.2 2.1 16 hr Lipoalone 7.8 55.7 15.2 18.5 100 nM 2302 8.4 57.3 14.3 17.3  10 nM 50 10.453.2 15.7 17.7 100 nM 50 27.7 31.3 18.1 19.6  10 nM 472 13.3 50.2 15.817.5 100 nM 472 30.7 31.9 16.4 18.0

In addition, over 50 phosphorothioate AS-ODNs complementary to the humanEphB4 coding sequences were synthesized and tested for their ability toinhibit EphB4 expression in 293 cells transiently transfected with fulllength EphB4 expression plasmid. FIG. 43A shows a representative sampleof the effect of some of these AS-ODNs on EphB4 expression. Note thatexpression is totally abrogated with AS-10, while AS-11 has only a minoreffect. The effect on cell viability in SCC15 cells was most marked withAS-ODNs that are most effective in inhibiting EphB4 expression as shownin FIG. 43B. The IC₅₀ for AS-10 was approximately 1 μM, while even 10 μMAS-11 was not sufficient to attain 50% reduction of viability. When theeffect that AS-10 had on the cell cycle was investigated, it was foundthat the sub G0 fraction increased from 1.9% to 10.5% compared tonon-treated cells, indicative of apoptosis (FIG. 43C).

E. EphB4 Regulates Cell Migration

We next wished to determine if EphB4 participates in the migration ofHNSCC. Involvement in migration may have implications for growth andmetastasis. Migration was assessed using the wound-healing/scrape assay.Confluent SCC15 and SCC25 cultures were wounded by a single scrape witha sterile plastic Pasteur pipette, which left a 3 mm band with clearlydefined borders. Migration of cells into the cleared area in thepresence of test compounds was evaluated and quantitated after 24, 48and 72 hr. Cell migration was markedly diminished in response to AS-10that block EphB4 expression while the inactive compounds, AS-1 andscrambled ODN had little to no effect as shown in FIG. 43D. Inhibitionof migration with AS-10 was also shown using the Boyden double chamberassay (FIG. 43E).

F. EphB4 AS-10 In Vivo Anti-Tumor Activity

The effect of EphB4 AS-10, which reduces cell viability and motility,was determined in SCC15 tumor xenografts in Balb/C nude mice. Dailytreatment of mice with 20 mg/kg AS-10, sense ODN or equal volume of PBSby I.P. injection was started the day following tumor cell implantation.Growth of tumors in mice receiving AS-10 was significantly retardedcompared to mice receiving either sense ODN or PBS diluent alone (FIG.44). Non-specific effects attributable to ODN were not observed, asthere was no difference between the sense ODN treated and PBS treatedgroups.

G. Materials and Methods

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

Example 6 Ephrin B2 Expression in Kaposi's Sarcoma is Induced by HumanHerpesvirus Type 8: Phenotype Switch from Venous to Arterial Endothelium

Kaposi's Sarcoma (KS) manifests as a multifocal angioproliferativedisease, most commonly of the skin and mucus membranes, with subsequentspread to visceral organs (1) Hallmarks of the disease are angiogenesis,edema, infiltration of lymphomononuclear cells and growth ofspindle-shaped tumor cells. Pathologically, established lesions exhibitan extensive vascular network of slit-like spaces. The KS vascularnetwork is distinct from normal vessels in the lack of basementmembranes and the abnormal spindle shaped endothelial cell (tumor cell)lining these vessels. Defective vasculature results in an accumulationof the blood components including albumin, red and mononuclear cells inthe lesions (1). The KS tumor is endothelial in origin; the tumor cellsexpress many endothelial markers, including lectin binding sites forUlex europeaus agglutinin-1 (UEA-1), CD34, EN-4, PAL-E (2) and theendothelial cell specific tyrosine kinase receptors, VEGFR-1 (Flt-1),VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), Tie-1 and Tie-2 (3, RM & PSGunpublished data). KS cells co-express lymphatic endothelial cellrelated proteins including LYVE and podoplanin (4).

The herpesvirus HHV-8 is considered the etiologic agent for the disease.In 1994 sequences of this new herpes virus were identified in KS tumortissue (5), and subsequent molecular-epidemiology studies have shownthat nearly all KS tumors contain viral genome. Sero-epidemiologystudies show that HIV infected patients with KS have the highestprevalence of HHV-8 and secondly that those with HIV infection but no KShave increased risk of developement of KS over the ensuing years if theyare also seropositive for HHV-8 (6). Direct evidence for the role ofHHV-8 in KS is the transformation of bone marrow endothelial cells afterinfection with HHV-8 (7). A number of HHV-8 encoded genes couldcontribute to cellular transformation (reviewed in 8). However, the mostevidence has accumulated for the G-protein coupled receptor (vGPCR) inthis role (9).

We investigated whether KS tumor cells are derived from arterial orvenous endothelium. In addition, we investigated whether HHV-8 has aneffect on expression of arterial or venous markers in a model of KS. KStumor cells were found to express the ephrin B2 arterial marker.Further, ephrin B2 expression was induced by HHV-8 vGPCR in KS andendothelial cell lines. Ephrin B2 is a potential target for treatment ofKS because inhibition of ephrin B2 expression or signaling wasdetrimental to KS cell viability and function.

A. KS Tumors Express Ephrin B2, but not EphB4

The highly vascular nature of KS lesions and the probable endothelialcell origin of the tumor cells prompted investigation of expression ofEphB4 and ephrin B2 which are markers for venous and arterialendothelial cells, respectively. Ephrin B2, but not EphB4 transcriptswere detected in tumor cells of KS biopsies by in situ hybridization(FIG. 45A). Comparison of the positive signal with ephrin B2 antisenseprobe and tumor cells as shown by H&E staining shows that ephrin B2expression is limited to the areas of the biopsy that contain tumorcells. The lack of signal in KS with EphB4 antisense probe is not due toa defect in the probe, as it detected transcripts in squamous cellcarcinoma, which we have shown expresses this protein (18). Additionalevidence for the expression of ephrin B2 in KS tumor tissue is affordedby the localization of EphB4/Fc signal to tumor cells, detected by FITCconjugated anti human Fc antibody. Because ephrin B2 is the only ligandfor EphB4 this reagent is specific for the expression of ephrin B2 (FIG.45B, left). An adjacent section treated only with the secondary reagentshows no specific signal. Two-color confocal microscopy demonstrated thepresence of the HHV-8 latency protein, LANA1 in the ephrin B2 positivecells (FIG. 45C, left), indicating that it is the tumor cells, not tumorvessels, which are expressing this arterial marker. Staining of tumorbiopsy with PECAM-1 antibody revealed the highly vascular nature of thistumor (FIG. 45C, right). A pilot study of the prevalence of this patternof ephrin B2 and EphB4 expression on KS biopsies was conducted by RT-PCRanalysis. All six samples were positive for ephrin B2, while only 2 wereweakly positive for EphB4 (data not shown).

B. Infection of Venous Endothelial Cells with HHV-8 Causes a PhenotypeSwitch to Arterial Markers

We next asked whether HHV-8, the presumed etiologic agent for KS, coulditself induce expression of ephrin B2 and repress EphB4 expression inendothelial cells. Co-culture of HUVEC and BC-1 lymphoma cells, whichare productively infected with HHV-8, results in effective infection ofthe endothelial cells (16). The attached monolayers of endothelial cellsremaining after extensive washing were examined for ephrin B2 and EphB4by RT-PCR and immunofluorescence. HUVEC express EphB4 venous markerstrongly at the RNA level, but not ephrin B2 (FIG. 46B). In contrast,HHV-8 infected cultures (HUVEC/BC-1 and HUVEC/BC-3) express ephrin B2,while EphB4 transcripts are almost absent.

Immunofluorescence analysis of cultures of HUVEC and HUVEC/HHV-8 forartery/vein markers and viral proteins was undertaken to determinewhether changes in protein expression mirrored that seen in the RNA. Inaddition, cellular localization of the proteins could be determined.Consistent with the RT-PCR data HUVEC are ephrin B2 negative and EphB4positive (FIG. 46A(a & m)). As expected they do not express any HHV-8latency associated nuclear antigen (LANA1) (FIG. 46A(b, n)). Co-cultureof BC-1 cells, which are productively infected with HHV-8, resulted ininfection of HUVEC as shown by presence of viral proteins LANA1 andORF59 (FIG. 46A(f, r)). HHV-8 infected HUVEC now express ephrin B2 butnot EphB4 (FIG. 46A(e, q, u), respectively). Expression of ephrin B2 andLANA1 co-cluster as shown by yellow signal in the merged image (FIG.46A(h)). HHV-8 infected HUVEC positive for ephrin B2 and negative forEph B4 also express the arterial marker CD148 (19) (FIG. 46A (j, v)).Expression of ephrin B2 and CD148 co-cluster as shown by yellow signalin the merged image (FIG. 46A(l)). Uninfected HUVEC expressing Eph B4were negative for CD148 (not shown).

C. HHV-8 vGPCR Induces Ephrin B2 Expression

To test whether individual viral proteins could induce the expression ofephrin B2 seen with the whole virus KS-SLK cells were stably transfectedwith HHV-8 LANA, or LANAΔ440 or vGPCR. Western Blot of stable clonesrevealed a five-fold induction of ephrin B2 in KS-SLK transfected withvGPCR compared to SLK-LANA or SLK-LANAΔ440 (FIG. 47A). SLK transfectedwith vector alone (pCEFL) was used as a control. SLK-vGPCR and SLK-pCEFLcells were also examined for ephrin B2 and Eph B4 expression byimmunofluorescence in transiently transfected KS-SLK cells. FIG. 47Bshows higher expression of ephrin B2 in the SLK-vGPCR cells compared toSLK-pCEFL. No changes in Eph B4 were observed in SLK-vGPCR compared toSLK-pCEFL. This clearly demonstrates that SLK-vGPCR cells expressed highlevels of ephrin B2 compared to SLK-pCEFL cells. This suggests thatvGPCR of HHV-8 is directly involved in the induction of Ephrin B2 andthe arterial phenotype switch in KS. Since we had shown that HHV-8induced expression of ephrin B2 in HUVEC, we next asked if this could bemediated by a transcriptional effect. Ephrin B2 5′-flankingDNA-luciferase reporter plasmids were constructed as described in theMaterials and Methods and transiently transfected into HUVECs. Ephrin B25′-flanking DNA sequences -2491/-11 have minimal activity in HUVEC cells(FIG. 47C). This is consistent with ephrin B2 being an arterial, notvenous marker. However, we have noted that HUVEC in culture do expresssome ephrin B2 at the RNA level. Cotransfection of HHV-8 vGPCR inducesephrin B2 transcription approximately 10-fold compared to the controlexpression vector pCEFL. Roughly equal induction was seen with ephrin B2sequences -2491/-11, -1242/-11, or -577/-11, which indicates thatelements between -577 and -11 are sufficient to mediate the response tovGPCR, although maximal activity is seen with the -1242/-11 luciferaseconstruct.

D. Expression of Ephrin B2 is Regulated by VEGF and VEGF-C

We next asked whether known KS growth factors could be involved in thevGPCR-mediated induction of ephrin B2 expression. SLK-vGPCR cells weretreated with neutralizing antibodies to oncostatin-M, IL-6, IL-8, VEGFor VEGF-C for 36 hr. FIG. 48A shows that neutralization of VEGFcompletely blocked expression of ephrin B2 in SLK-vGPCR cells. A lesser,but significant decrease in ephrin B2 was seen neutralization of VEGF-Cand IL-8. No appreciable effect was seen with neutralization ofoncostatin-M or IL-6. To verify that VEGF and VEGF-C are integral to theinduction of ephrin B2 expression we treated HUVEC with VEGF, VEGF-C orEGF. HUVECs were grown in EBM-2 media containing 5% FBS with twodifferent concentration of individual growth factor (10 ng, 100 ng/ml)for 48 h. Only VEGF-A or VEGF-C induced ephrin B2 expression in a dosedependent manner (FIG. 48B). In contrast, EGF had no effect onexpression of ephrin B2.

E. Ephrin B2 siRNA Inhibits the Expression of Ephrin B2 in KS

Three ephrin B2 siRNA were synthesized as described in the methodssection. KS-SLK cells were transfected with siRNA and 48 h later ephrinB2 expression was determined by Western Blot. Ephrin B2 siRNAs 137 or254 inhibited about 70% of ephrin B2 expression compared to controlsiRNA such as siRNA Eph B4 50 or siRNA GFP. Ephrin B2 63 siRNA was lesseffective than the above two siRNA Ephrin B2 (FIG. 49A).

F. Ephrin B2 is Necessary for Full KS and EC Viability, Cord Formationand In Vivo Angiogenesis Activities

The most effective ephrin B2 siRNA (254) was then used to determinewhether inhibiting expression of ephrin B2 has any effect on the growthof KS-SLK or HUVEC cells. The viability of KS-SLK cells was decreased bythe same siRNAs that inhibited ephrin B2 protein levels (FIG. 49B).KS-SLK express high levels of ephrin B2 and this result showsmaintenance of ephrin B2 expression is integral to cell viability inthis setting. HUVECs do not express ephrin B2, except when stimulated byVEGF as shown in FIG. 48B. Ephrin B2 siRNA 264 dramatically reducedgrowth of HUVECs cultured with VEGF as the sole growth factor. Incontrast, no significant effect was seen when HUVECs were cultured withIGF, EGF and bFGF. As a control, EphB4 siRNA 50 had no detrimentaleffect on HUVECs in either culture condition (FIG. 49C). In addition toinhibition of viability of KS and primary endothelial cells, EphB4-ECDinhibits cord formation in HUVEC and KS-SLK and in vivo angiogenesis inthe Matrigel™ plug assay (FIG. 50).

G. Methods and Materials

A detailed description of the materials and methods for this example maybe found in U.S. Patent Publication No. 20050084873.

Example 7 Expression of EphB4 in Bladder Cancer: a Candidate Target forTherapy

FIG. 51 shows expression of EPHB4 in bladder cancer cell lines (A), andregulation of EPHB4 expression by EGFR signaling pathway (B).

FIG. 52 shows that transfection of p53 inhibit the expression of EPHB4in 5637 cell.

FIG. 53 shows growth inhibition of bladder cancer cell line (5637) upontreatment with EPHB4 siRNA 472.

FIG. 54 shows results on apoptosis study of 5637 cells transfected withEPHB4 siRNA 472.

FIG. 55 shows effects of EPHB4 antisense probes on cell migration. 5637cells were treated with EPHB4AS10 (10 μM).

FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637 cells weretransfected with siRNA 472 or control siRNA.

Example 8 Inhibition of EphB4 Gene Expression by EphB4 Antisense Probesand RNAi Probes

Cell lines expressing EphB4 were treated with the syntheticphosphorothioate modified oligonucleotides and harvested after 24 hr.Cell lysates were prepared and probed by western blot analysis forrelative amounts of EphB4 compared to untreated control cells.

Studies on inhibition of cell proliferation were done in HNSCC celllines characterized to express EphB4. Loss of cell viability was shownupon knock-down of EphB4 expression. Cells were treated in vitro andcultured in 48-well plates, seeded with 10 thousand cells per well. Testcompounds were added and the cell viability was tested on day 3. Theresults on EphB4 antisense probes were summarized below in Table 6. Theresults on EphB4 RNAi probes were summarized below in Table 7.

A detailed description of the antisense and siRNA constructs for thisexample may be found in U.S. Patent Publication No. 20050084873.

Example 9 Inhibition of Ephrin B2 Gene Expression by Ephrin B2 AntisenseProbes and RNAi Probes

KS SLK, a cell line expressing endogenous high level of ephrin B2. Cellviability was tested using fixed dose of each oligonuceotide (5 uM).Gene expression downregulation was done using cell line 293 engineeredto stably express full-length ephrin B2. KS SLK expressing EphrinB2 werealso used to test the viability in response to RNAi probes tested at thefixed dose of 50 nM. Protein expression levels were measured using 293cells stably expressing full-length EphrinB2, in cell lysates after 24hr treatment with fixed 50 nM of RNAi probes.

The results on Ephrin B2 antisense probes were summarized below in Table8. The results on Ephrin B2 RNAi probes were summarized below in Table9.

A detailed description of the antisense and siRNA constructs for thisexample may be found in U.S. Patent Publication No. 20050084873.

Example 10 EphB4 Antibodies Inhibit Tumor Growth

FIG. 57 shows results on comparison of EphB4 monoclonal antibodies byG250 and in Pull-down assay.

FIG. 58 shows that EphB4 antibodies, in the presence of matrigel andgrowth factors, inhibit the in vivo tumor growth of SCC15 cells.

BaIbC nude mice were injected subcutaneously with 2.5×10⁶ viable tumorcells SCC15 is a head and neck squamous cell carcinoma line. Tumors wereinitiated in nu/nu mice by injecting 2.5-5×10⁶ cells premixed withmatrigel and Growth factors, and Ab's subcutaneously to initiate tumorxenografts. Mice were opened 14 days after injections. SCC15 is a headand neck squamous cell carcinoma line, B16 is a melanoma cell line, andMCF-7 is a breast carcinoma line. The responses of tumors to thesetreatments were compared to control treated mice, which receive PBSinjections. Animals were observed daily for tumor growth andsubcutaneous tumors were measured using a caliper every 2 days.Antibodies #1 and #23 showed significant regression of SCC15 tumor sizecompared to control, especially with no additional growth factor added.

FIG. 59 shows that EphB4 antibodies cause apoptosis, necrosis anddecreased angiogenesis in SCC15, head and neck carcinoma tumor type.

Angiogenesis was assessed by CD-31 immunohistochemistry. Tumor tissuesections from treated and untreated mice were stained for CD31.Apoptosis was assessed by immunohistochemical TUNNEL, and proliferationby BrdU assay. Following surgical removal, tumors were immediatelysliced into 2 mm serial sections and embedded in paraffin using standardprocedures. Paraffin embedded tissue were sectioned at 5 μm, the waxremoved and the tissue rehydrated. The rehydrated tissues were microwaveirradiated in antigen retreival solution. Slides were rinsed in PBS, andTUNNEL reaction mixture (Terminal deoxynucleotidyl transferase andflourescein labeled nucleotide solution), and BrdU were added in ahumidity chamber completely shielded from light. The TUNNEL and BrdUreaction mixture were then removed, slides were rinsed andanti-flourescein antibody conjugated with horseradish peroxidase wasadded. After incubation and rinsing, 3, 3′ diaminobenzidine was added.Masson's Trichrome and Hematoxylin and Eosin were also used to stain theslides to visualize morphology. Masson's Trichrome allows to visualizenecrosis and fibrosis. The tumor gets blood support from tumor/skin,muscle boundary. As tumor grows, inner regions get depleted ofnutrients. This leads to necrosis (cell death), preferably at the tumorcenter. After cells die, (tumor) tissue gets replaced with fibroblastictissue. Slides were visualized under 20-fold magnification with digitalimages acquired. A different morphology was obtained on SCC tumors witheach antibody administered. Ab #1 showed an increase in necrosis andfibrosis but not apoptosis. Ab #23 showed an increase in apoptosis,necrosis and fibrosis and a decrease in vessel infiltration. Ab #35showed an increase in necrosis and fibrosis, and a small increase inapoptosis and a decrease in vessel infiltration. Ab #79 showed a largeincrease in apoptosis, and necrossis and fibrosis. Ab #91 showed nochange in apoptosis but an increase in proliferation. And Ab #138 showedan increase in apoptosis, necrosis, fibrosis and a decrease inproliferation and vessel infiltration. Tumors treated with control PBSdisplayed abundant tumor density and a robust angiogenic response.Tumors treated with EphB4 antibodies displayed a decrease in tumor celldensity and a marked inhibition of tumor angiogenesis in regions withviable tumor cells, as well as tumor necrosis and apoptosis.

FIG. 60 shows that systemic administration of antibodies on xenograftsleads to tumor regression in SCC15 tumor xenografts.

Alternate day treatment with EphB4 monoclonal antibody or an equalvolume of PBS as control were initiated on day 4, after the tumors haveestablished, and continued for 14 days. Systemic administration wasadministered either IP or SC with no significant difference. All theexperiments were carried out in a double-blind manner to eliminateinvestigator bias. Mice were sacrificed at the conclusion of the twoweek treatment period. Tumors were harvested immediately postmortem andfixed and processed for immunohistochemistry. EphB4 antibodies 40 mg perkg body weight were administered. Treatment with EphB4 antibodysignificantly inhibited human SCC tumor growth compared withcontrol-treated mice (p<0.05). Treatment with EphB4 antibodysignificantly inhibited tumor weight compared with control-treated mice(p<0.05).

Example 11 HSA-EphB4 Ectodomain Fusion and PEG-Modified EphB4 EctodomainA. Generation of HSA-EphB4 Ectodomain Fusion

Human serum albumin fragment in XbaI-NotI form was PCR-amplified out forcreating a fusion with GCF2, and TA-cloned into pEF6. In the next step,the resulting vector was cut with Xba I (partial digestion) and the HSAfragment (1.8 kb) was cloned into Xba I site of pEF6-GCF2-Xba to createfusion expression vector. The resulting vector had a point mutation C toT leading to Thr to Ile substitution in position 4 of the matureprotein. It was called pEF6-GCF2-HSAmut. In the next cloning step, themutation was removed by substituting wild type KpnI fragment frompEF6-GCF2-IF (containing piece of the vector and N-terminal part ofGCF2) for the mutated one, this final vector was called pEF6-GCF2. TheDNA sequence of pEF6-GCF2 was confirmed.

The predicted amino acid of the HSA-EphB4 precursor protein was asfollows (SEQ ID NO:18):

MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAVLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

The predicted amino acid sequence of the mature form of the HSA-EphB4protein was as follows (SEQ ID NO:19):

LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAVLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLThe nucleic acid sequence of the pEF6-GCF2 plasmid was as follows (SEQID NO: 20):

aatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactaggcttttgcaaaaagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgcctcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttatttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggaattagcttggtactaatacgactcactatagggagacccaagctggctaggtaagcttggtaccgagctcggatccactagtccagtgtggtggaattgcccttCAAGCTTGCCGCCACCATGGAGCTCCGGGTGCTGCTCTGCTGGGCTTCGTTGGCCGCAGCTTTGGAAGAGACCCTGCTGAACACAAAATTGGAAACTGCTGATCTGAAGTGGGTGACATTCCCTCAGGTGGACGGGCAGTGGGAGGAACTGAGCGGCCTGGATGAGGAACAGCACAGCGTGCGCACCTACGAAGTGTGTGACGTGCAGCGTGCCCCGGGCCAGGCCCACTGGCTTCGCACAGGTTGGGTCCCACGGCGGGGCGCCGTCCACGTGTACGCCACGCTGCGCTTCACCATGCTCGAGTGCCTGTCCCTGCCTCGGGCTGGGCGCTCCTGCAAGGAGACCTTCACCGTCTTCTACTATGAGAGCGATGCGGACACGGCCACGGCCCTCACGCCAGCCTGGATGGAGAACCCCTACATCAAGGTGGACACGGTGGCCGCGGAGCATCTCACCCGGAAGCGCCCTGGGGCCGAGGCCACCGGGAAGGTGAATGTCAAGACGCTGCGCCTGGGACCGCTCAGCAAGGCTGGCTTCTACCTGGCCTTCCAGGACCAGGGTGCCTGCATGGCCCTGCTATCCCTGCACCTCTTCTACAAAAAGTGCGCCCAGCTGACTGTGAACCTGACTCGATTCCCGGAGACTGTGCCTCGGGAGCTGGTTGTGCCCGTGGCCGGTAGCTGCGTGGTGGATGCCGTCCCCGCCCCTGGCCCCAGCCCCAGCCTCTACTGCCGTGAGGATGGCCAGTGGGCCGAACAGCCGGTCACGGGCTGCAGCTGTGCTCCGGGGTTCGAGGCAGCTGAGGGGAACACCAAGTGCCGAGCCTGTGCCCAGGGCACCTTCAAGCCCCTGTCAGGAGAAGGGTCCTGCCAGCCATGCCCAGCCAATAGCCACTCTAACACCATTGGATCAGCCGTCTGCCAGTGCCGCGTCGGGTACTTCCGGGCACGCACAGACCCCCGGGGTGCACCCTGCACCACCCCTCCTTCGGCTCCGCGGAGCGTGGTTTCCCGCCTGAACGGCTCCTCCCTGCACCTGGAATGGAGTGCCCCCCTGGAGTCTGGTGGCCGAGAGGACCTCACCTACGCCCTCCGCTGCCGGGAGTGTCGACCCGGAGGCTCCTGTGCGCCCTGCGGGGGAGACCTGACTTTTGACCCCGGCCCCCGGGACCTGGTGGAGCCCTGGGTGGTGGTTCGAGGGCTACGTCCTGACTTCACCTATACCTTTGAGGTCACTGCATTGAACGGGGTATCCTCCTTAGCCACGGGGCCCGTCCCATTTGAGCCTGTCAATGTCACCACTGACCGAGAGGTACCTCCTGCAGTGTCTGACATCCGGGTGACGCGGTCCTCACCCAGCAGCTTGAGCCTGGCCTGGGCTGTTCCCCGGGCACCCAGTGGGGCTGTGCTGGACTACGAGGTCAAATACCATGAGAAGGGCGCCGAGGGTCCCAGCAGCGTGCGGTTCCTGAAGACGTCAGAAAACCGGGCAGAGCTGCGGGGGCTGAAGCGGGGAGCCAGCTACCTGGTGCAGGTACGGGCGCGCTCTGAGGCCGGCTACGGGCCCTTCGGCCAGGAACATCACAGCCAGACCCAACTGGATGAGAGCGAGGGCTGGCGGGAGCAGtctagaGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTAGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAATGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTGGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAGGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAACTGTGAGCTTTTTAAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACAAAATGCTGCACAGAGTCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATAAtagcggccgcttaagggcaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttggggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagcatttgtctcaagaagaatccaccctcattgaaagagcaacggctacaatcaacagcatccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacggccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgtgcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctgacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctgcggacggtgtcgacaggtgcttctcgatctgcatcctgggatcaaagcgatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggctaagcacttcgtggccgaggagcaggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggttgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttc

B. Cell Culture and Transfections:

The human embryonic kidney cell line, 293T cells, was maintained in DMEMwith 10% dialyzed fetal calf serum and 1%penicillin/streptomycin/neomycin antibiotics. Cells were maintained at37° C. in a humidified atmosphere of 5% CO₂/95% air.Transfections of plasmids encoding EphB4 ectodomain, fragments thereof,and EphB4-HSA fusions were performed using Lipofectamine 2000 reagent(Invitrogen) according to suggested protocol. One day beforetransfections, 293T cells were seeded at a high density to reach 80%confluence at the time of transfection. Plasmid DNA and Lipofectaminereagent at 1:3 ratio were diluted in Opti-MEM I reduced serum medium(Invitrogen) for 5 min and mixed together to form DNA-Lipofectaminecomplex. For, each 10 cm culture dish, 10 μg of plasmid DNA was used.After 20 min, the above complex was added directly to cells in culturemedium. After 16 hours of transfection, medium was aspirated, washedonce with serum free DMEM and replaced with serum free DMEM. Secretedproteins were harvested after 48 hours by collecting conditional medium.Conditional medium was clarified by centrifugation at 10,000 g for 20min and filtered through 0.2μ filter and used for purification.

C. Chromatographic Separation of EphB4 Ectodomain and EphB4Ectodomain-HSA Fusion Protein

The EphB4 ectodomain fused to HSA was purified as follows: 700 ml ofmedia was harvested from transiently transfected 293 cells grown inserum free media and concentrated up to final volume of 120 ml.Membrane: (Omega, 76 mm), 50 kDa C/O. After concentration, pH of thesample was adjusted by adding 6 ml of 1M NaAc, pH 5.5. Then sample wasdialyzed against starting buffer (SB): 20 mM NaAc, 20 mM NaCl, pH 5.5for O/N. 5 ml of SP-Sepharose was equilibrated with SB and sample wasloaded. Washing: 100 ml of SB. Elution by NaCl: 12 ml/fraction andincrement of 20 mM. Most of the EphrinB2 binding activity eluted in the100 mM and 120 mM fractions.

Fractions, active in EphrinB2 binding assay (See SP chromatography,fractions #100-120 mM) were used in second step of purification onQ-column. Pulled fractions were dialyzed against starting buffer #2(SB2): 20 mM Tris-HCl, 20 mM NaCl, pH 8 for O/N and loaded onto 2 ml ofQ-Sepharose. After washing with 20 ml of SB2, absorbed protein waseluted by NaCl: 3 ml/fraction with a concentration increment of 25 mM.Obtained fractions were analyzed by PAGE and in Ephrin-B2 binding assay.The 200 mM and 225 mM fractions were found to contain the most proteinand the most B2 binding activity.

Soluble EphB4 ectodomain protein was purified as follows: 300 ml ofconditional medium (see: Cell culture and transfections) wereconcentrated up to final volume of 100 ml, using ultrafiltrationmembrane with 30 kDa C/O. After concentration, pH of the sample wasadjusted by adding 5 ml of 1 M Na-Acetate, pH 5.5. Then sample wasdialyzed against starting buffer (StB): 20 mM Na-Acetate, 20 mM NaCl, pH5.5 for O/N. 5 ml of SP-Sepharose was equilibrated with StB and samplewas loaded. After washing the column with 20 ml of StB, absorbedproteins were eluted by linear gradient of concentration of NaCl (20-250mM and total elution volume of 20 column's volumes). Purity of theproteins was analyzed by PAGE.

D. Biotinylation of sB4 and sB4-HSA Fusion Protein.

Both soluble EphB4 ectodomain protein (sB4) and EphB4 ectodomain fusedto HSA (HSA-sB4) were biotin labeled through carbohydrate chains usingsodium meta-periodate as an oxidant and EZ-Link Biotin Hydrazide(PIERCE, Cat. #21339) according to manufacture's protocol. The in vitrostability of the biotinylated sB4 protein was tested by incubating2.0×10⁻⁹ with 40 μL of mouse serum at 37° C. for 0, 0.5, 1, 2 and 3days. Two μL of magnetic beads and B2-AP was added for an extra hour atroom temperature. After washing twice with buffer, pnPP was added for 1hour. Biotinylated sB4 protein was found to very stable over three days,with less than 10% of the B2 binding activity being lost over that time.

E. Ephrin-B2 Binding Properties of B4-HSA

To test whether the B4-HSA fusion property retained the ability of theEphB4 extracellular domain to bind to EphrinB2, the ability of thepurified B4-HSA fusion was compared to that of GCF2F, GCF2, GC, CF andB4-Fc fusion, which comprises the extracellular domain of B4 fused tohIgG1 Fc as described in Example 1. Biotinylated or His-tag proteinsamples were inoculated with the corresponding affinity magnetic beadsand B2-AP for an hour at room temperature, before addition of PnPP.Results of binding assays are shown on FIG. 67. B4-HSA was found toretain most of its binding activity towards EphrinB2. Surprisingly, theB4-HSA protein was superior to the B4-Fc fusion in binding to EphrinB2.

An EphB4 ectodomain fusion to the C-terminus of HSA was also generated,and found to retain the ability to bind to EphrinB2 and was found tohave enhanced stability in vivo over the EphB4 ectodomain.

F. Stability of B4-HSA vs. sB4 in Mice

The stability of the purified biotinylated sB4 and sB4-HSA were assayedin vivo. Each of the proteins were intravenously injected into the tailof mice in the amount of 0.5 nmoles per mouse. Blood from the eye ofeach mouse was taken in time frames of 15 min (0 days), 1, 2, 3 and 6days. 10 ml of obtained serum was used in binding assay withEphrin-B2-Alkaline Phosphatase fusion protein and Streptavidin-coatedmagnetic beads as a solid phase. The stability of the two proteins isshown on FIG. 68. sB4-HSA was found to have superior stability relativeto sB4. For example, one day after injection, the levels of sB4-HSA inthe blood of the mice were 5-fold greater than those of sB4.

G. PEGylation of Biotinylated sB4

Prior to PEGylation, biotinylated sB4 protein generated as describedabove was concentrated up to final concentration of 2 mg/ml using a 30kDa MWCO ultra membrane. Sample was dialyzed O/N against couplingbuffer: 30 mM phosphate, 75 mM NaCl, pH 8.00. Coupling to PEG wasperformed at 4° C. for 18 hours in 10 fold molar excess of reactivelinear PEG unless otherwise indicated. The reactive PEG used wasPEG-succinimidyl propionate, having a molecular weight of about 20 kda.Coupling to PEG may be similarly performed using branches PEGS, such asof 10 kDa, 20 kDa or 40 kDa. Other linear PEG molecules of 10 or 40 kDamay also be used.

After PEGylation, the protein sample containing EphB4 ectodomain wasdialyzed against StB O/N. Three ml of SP-Sepharose was equilibrated withStB and sample was loaded. Washing and elution of absorbed proteins wasperformed as above (see: Purification of soluble EphB4 ectodomain andits fusion to HSA) with just one modification: total elution volume was40 volumes of column. FIG. 69 shows chromatographic separation of PEGderivatives of EphB4 protein on SP-Sepharose columns. Purity of thePEG-modified EphB4 protein was analyzed by SDS-PAGE.

Double modified (PEGylated Biotinylated) sB4 was used on ion-exchangechromatography to separate non-PEGylated, mono-PEGylated andpoly-PEGylated proteins from each other. Pegylated sample was dialyzedO/N against 20 mM Na-acetate, 20 mM NaCl, pH 5.5 and loaded onto 2 ml ofSP-Sepharose. After washing with 10 ml of buffer, absorbed proteins wereseparated by gradual elution of NaCl: 3 ml/fraction and increment of 25mM NaCl. Obtained fractions were analyzed by PAGE and in Ephrin-B2binding assay.

H. Effect of PEGylation Conditions on sB4 Binding to EphrinB2

The effects of pegylating biotinylated sB4 under different pH conditionswas determined. sB4 was pegylated at pH 6, 7 or 8, and the pegylatedproducts were tested for binding to EphrinB2 as shown in FIG. 69.Ephrin2B binding activity was retained when PEGylation was performed atpH 6 and pH 7, but was partially lost at pH 8.

Additional combinations of parameters were tested, includingtemperature, pH and molar ratio of pegylation agent to sB4 protein, andthe ability of the products of the pegylation reaction to bind toEphrin-B2. The results of the optimization experiment are shown in FIG.70. These results confirm the gradual decrease in B2 binding activity atbasic pH.

I. Purification of Pegylated sB4 Species

Biotinylated sB4 protein was concentrated up to final concentration of 2mg/ml using a 30 kDa MWCO ultra membrane. Sample was dialyzed O/Nagainst coupling buffer: 30 mM phosphate, 75 mM NaCl, pH 8.00. Couplingto PEG was performed at 4° C. for 18 hours in 10 fold molar excess ofreactive PEG. Double modified (PEGylated Biotinylated) sB4 was used onion-exchange chromatography to separate non-PEGylated, mono-PEGylatedand poly-PEGylated proteins from each other. Sample was dialyzed for O/Nagainst 20 mM Na-Acetate, 20 mM NaCl, pH 5.5 and loaded onto 2 ml ofSP-Sepharose. After washing with 10 ml of buffer, absorbed proteins wereseparated by gradual elution of NaCl: 3 refraction and increment of 25mM NaCl. Obtained fractions were analyzed by PAGE as shown in FIG. 71.Fractions 1, 2 and 3 were found to correspond to polypegylated,monopegylated and unpegylated biotinylated sB4.

J. In Vitro Properties of PEGylated EphB4 Derivatives

Fractions 1, 2 and 3 of biotinylated and PEGylated sB4 from the SPcolumn purification, corresponding to polypegylated, monopegylated andunpegylated biotinylated sB4, were tested for their ability to bindEphrinB2 using the standard assay. Results of this experiment are shownon FIG. 72. The order of binding activity was found to beUnpegylated>monopegylated>polypegylated.

The fractions were also tested for their stability in vitro. Thefractions were tested for retention of EphrinB2 binding activity afterincubation in mouse serum at 37° C. for three days. The results of thisexperiment are shown in FIG. 73. The order of in vitro stability wasfound to be monopegylated>unpegylated>polypegylated.

K. In Vivo Stability Analysis of PEGylated Derivatives of EphB4Ectodomain in Mice

Fractions 1, 2 and 3 of biotinylated and PEGylated sB4 from the SPcolumn purification, corresponding to polypegylated, monopegylated andunpegylated biotinylated sB4, were introduced by intravenous injectioninto mice in the amount of 0.5 nMoles/mouse. Blood from each mouse wastaken in time frame of 10 min, 1, 2 and 3 days. 10 ml of obtained serumwas used in binding assay with Ephrin-B2-Alkaline Phosphatase fusionprotein and Streptavidin-coated magnetic beads as a solid phase.Signals, obtained at 10 min were taken as 100%. The two mice for eachprotein were of a different strain. Results are shown in FIG. 74.Pegylation was found to increase the stability of EphB4 in vivo relativeto unpegylated EphB4.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. An isolated soluble polypeptide comprising an amino acid sequence ofan extracellular domain of an EphB4 protein, wherein the polypeptide isa monomer and binds specifically to an Ephrin B2 polypeptide.
 2. Thepolypeptide of claim 1, comprising a globular domain of an EphB4 proteinor a sequence that is at least 90% identical to a globular domain ofEphB4.
 3. The polypeptide of claim 1, comprising a sequence at least 90%identical to residues 29-197 of the amino acid sequence defined by FIG.65 (SEQ ID NO:10).
 4. The polypeptide of claim 1, further comprising amodification that increases serum half-life.
 5. The polypeptide of claim4, wherein said modification comprises a polyethylene glycol group. 6.The polypeptide of claim 5, wherein said modification is a singlepolyethylene glycol group covalently bonded to the polypeptide.
 7. Thepolypeptide of claim 5, wherein said polypeptide is covalently bonded totwo polyethylene glycol groups.
 8. The polypeptide of claim 5, whereinsaid polypeptide is covalently bonded to multiple polyethylene glycolgroups.
 9. The polypeptide of claim 5, wherein said polyethylene glycolgroup has a molecular weight of from about 10 to about 40 kDa.
 10. Thepolypeptide of claim 5, wherein the polyethylene glycol group has amolecular weight of from about 30 to about 40 kDa.
 11. The polypeptideof claim 5, wherein said polyethylene glycol group is selected fromlinear PEG chains and branched PEG chains.
 12. The polypeptide of claim5, wherein said polyethylene glycol group is attached to a groupselected from the lysine side chains and the N-terminal amino group ofthe EphB4 polypeptide.
 13. The polypeptide of claim 4, wherein saidpolypeptide has a serum half-life in vivo at least 50% greater than thatof an unmodified EphB4 polypeptide.
 14. The polypeptide of claim 4,wherein said polypeptide has a serum half-life in vivo at least 100%greater than that of an unmodified EphB4 polypeptide.
 15. Thepolypeptide of claim 4, wherein the polypeptide is a fusion protein. 16.The polypeptide of claim 15, wherein the polypeptide comprises analbumin protein or fragments thereof.
 17. The polypeptide of claim 16,wherein said albumin protein is selected from a human serum albumin(HSA) and bovine serum albumin (BSA).
 18. The polypeptide of claim 16,wherein the albumin is a naturally occurring variant.
 19. Thepolypeptide of claim 1, wherein the polypeptide has one or moreactivities selected from: (a) inhibition of EphrinB2 activity; (b)inhibition of EphrinB2 kinase activity; (c) inhibition of theinteraction between EphB4 and EphrinB2; (d) inhibition of EphB4 kinaseactivity; (e) inhibition of clustering of Ephrin B2; and (f) inhibitionof clustering of EphB4.
 20. The polypeptide of claim 4, wherein thepolypeptide has enhanced in vivo stability relative to the unmodifiedwildtype polypeptide.
 21. A pharmaceutical composition comprising apolypeptide of claim 1, and a pharmaceutically acceptable carrier.
 22. Amethod of inhibiting signaling through Ephrin B2/EphB4 pathway in acell, comprising contacting the cell with an effective amount of apolypeptide of claim
 1. 23. A method of reducing the growth rate of atumor, comprising administering an amount of a polypeptide of claim 1,sufficient to reduce the growth rate of the tumor.
 24. A method fortreating a patient suffering from a cancer, comprising administering tothe patient a polypeptide of claim
 1. 25. A method of inhibitingangiogenesis, comprising contacting a cell with a polypeptide ofclaim
 1. 26. A method for treating a patient suffering from anangiogenesis-associated disease, comprising administering to the patienta polypeptide of claim
 1. 27. The polypeptide of claim 1, wherein thepolypeptide comprises one or more modified amino acid residues.
 28. Acosmetic composition comprising the polypeptide of claim 1, and apharmaceutically acceptable carrier.
 29. A method of reducing the growthrate of a tumor, comprising administering an amount of a polypeptideagent sufficient to reduce the growth rate of the tumor, wherein thepolypeptide agent is selected from: (a) a soluble polypeptide comprisingan amino acid sequence of an extracellular domain of an EphB4 protein,wherein the EphB4 polypeptide is a monomer and binds specifically to anEphrin B2 polypeptide; and (b) a soluble polypeptide comprising an aminoacid sequence of an extracellular domain of an Ephrin B2 protein,wherein the soluble Ephrin B2 polypeptide is a monomer and binds withhigh affinity to an EphB4 polypeptide.
 30. The method of claim 29,wherein the tumor comprises cells expressing a higher level of EphB4and/or EphrinB2 than noncancerous cells of a comparable tissue.
 31. Amethod for treating a patient suffering from a cancer, comprisingadministering to the patient a polypeptide agent selected from: (a) asoluble polypeptide comprising an amino acid sequence of anextracellular domain of an EphB4 protein, wherein the EphB4 polypeptideis a monomer and binds specifically to an Ephrin B2 polypeptide; and (b)a soluble polypeptide comprising an amino acid sequence of anextracellular domain of an Ephrin B2 protein, wherein the soluble EphrinB2 polypeptide is a monomer and binds with high affinity to an EphB4polypeptide.
 32. The method of claim 31, wherein the cancer comprisescancer cells expressing EphrinB2 and/or EphB4 at a higher level thannoncancerous cells of a comparable tissue.
 33. The method of claim 31,wherein the cancer is metastatic cancer.
 34. The method of claim 31,wherein the tumor is selected from colon carcinoma, breast tumor,mesothelioma, prostate tumor, squamous cell carcinoma, Kaposi sarcoma,and leukemia.
 35. The method of claim 31, wherein the cancer is anangiogenesis-dependent cancer.
 36. The method of claim 31, wherein thecancer is an angiogenesis-independent cancer.
 37. The method of claim31, wherein the polypeptide agent is a soluble polypeptide comprising anamino acid sequence of an extracellular domain of an Ephrin B2 protein,wherein the soluble Ephrin B2 polypeptide is a monomer and binds withhigh affinity to an EphB4 polypeptide and further comprises amodification that increases serum half-life.
 38. The method of claim 31,further including administering at least one additional anti-cancerchemotherapeutic agent that inhibits cancer cells in an additive orsynergistic manner with the polypeptide agent.
 39. A method ofinhibiting angiogenesis, comprising contacting a cell with an amount ofa polypeptide agent sufficient to inhibit angiogenesis, wherein thepolypeptide agent is selected from: (a) a soluble polypeptide comprisingan amino acid sequence of an extracellular domain of an EphB4 protein,wherein the EphB4 polypeptide is a monomer and binds specifically to anEphrin B2 polypeptide; and (b) a soluble polypeptide comprising an aminoacid sequence of an extracellular domain of an Ephrin B2 protein,wherein the soluble Ephrin B2 polypeptide is a monomer and binds withhigh affinity to an EphB4 polypeptide.
 40. A method for treating apatient suffering from an angiogenesis-associated disease, comprisingadministering to the patient a polypeptide agent selected from: (a) asoluble polypeptide comprising an amino acid sequence of anextracellular domain of an EphB4 protein, wherein the EphB4 polypeptideis a monomer and binds specifically to an Ephrin B2 polypeptide; and (b)a soluble polypeptide comprising an amino acid sequence of anextracellular domain of an Ephrin B2 protein, wherein the soluble EphrinB2 polypeptide is a monomer and binds with high affinity to an EphB4polypeptide. 41-47. (canceled)